KR100816737B1 - Micropropagation technique via somatic embryogenesis in oplopanax elatus - Google Patents

Abstract

A method for propagating Oplopanax elatus is provided to obtain a complete plantlet by inducing embryogenic callus from zygotic embryos of the Oplopanax elatus, which is hard to be propagated by seedling due to low seed yield and low fertility. A method for propagating Oplopanax elatus comprises the steps of: (a) culturing zygotic embryos of seeds of surface sterilized Oplopanax elatus to induce a somatic embryogenic tissue; (b) culturing the induced somatic embryogenic tissue to maintain and proliferate the embryogenic tissue to induce somatic embryos; (c) culturing the somatic embryos in an MS culture medium where 1.0-10.0 mg/L of gibberellic acid is included to induce a plantlet; and (d) transplanting the plantlet into an artificial bed soil to acclimatize the plantlet by keeping it in a greenhouse having the moisture of more than 90%, wherein the surface sterilization is done by washing the seeds of the Oplopanax elatus with Tween 20 added water, immersing them into ethanol, a Triton X-100 added NaClO solution and a HgCl2 solution in sequence to sterilize, and then washing the sterilized seeds with sterile distilled water.

Description

Micropropagation technique via somatic embryogenesis in Oplopanax elatus

Figure 1 is a photograph of somatic embryogenic tissue derived from mature seedlings.

Figure 2 is a photograph of somatic embryos induced from seed-derived embryonic tissues of the elder tree.

Figure 3 is a photograph of the somatic embryos of the elder tree matured to mature stages of several stages.

Figure 4 is a photograph of the step of growing cotyledon and contraction of the somatic embryos of the elm tree in the germination process.

5 is a photograph of germ somatic embryos germinated and re-divided into plants.

Figure 6 is a photograph of young plants re-differentiated from somatic embryos of young elm and then transplanted into artificial soil and purified.

The present invention relates to the in vitro propagation of the elm tree through somatic embryogenesis, and more particularly to the in vitro propagation method of the elm tree to induce a complete plant by inducing somatic embryos from the zygotic embryo of mature seeds. .

Somatic embryo induction is the most efficient technique of breeding technology of tissue culture, by applying tissue culture technology to artificially induce embryos in vitro and forming plants through a series of embryo development processes. . Somatic embryo induction technology was first observed in carrot cultured cells by Steward et al. In 1958. Citrus fruits (Ranga Swamy, 1958), Biota orientalis (Konar and Oberoi, 1965), Santalum album (Rao, 1965), Zamia integrifolia (Norstog, 1965) and others.

In 1985, the conifer species, the German spruce ( Picea Somatic embryos are induced in abies ), and there has been an explosive increase in the study of somatic embryo induction in trees, which has been reported so far in more than 150 species of trees (Dunstan et al. 1995). However, although somatic embryo technology is a breakthrough technology that can differentiate from single cells to complete plants, there are still many problems to be solved, the most difficult of which is to derive embryonic tissue from sections (Bodhipadma and Leung, 2002; Dai et al. 2004). The degree of induction of this embryogenic tissue is closely related to the juvenility of the tissue (Bonga 2004; Capuana and Debergh 1997; Chalupa 2000; Feher et al. 2003; Merkle et al. 1997, 1998, 2000, 2003), therefore, the optimization of culture conditions considering the stages of development of splicer embryos of immature seeds is one of the most important factors. Therefore, inducing embryogenic tissues by selecting more abundant tissues regardless of species is the first step in the application of somatic embryo induction technology (Raemakers et al. 1999; Sharp wt al. 1980; Vendrame et al. 2001 Vookova et al. 2003).

Studies on induction of somatic embryos on elm and elm have been developed by the authors, etc. (Moon and Youn, 1999; Moon et al., 2005). 1985) showed that somatic embryogenic cells were obtained from sections of fire tissue, but they did not achieve plant formation through induction, embryonic maturation and germination of somatic embryos.

Thus, there have been no reports of plant development through somatic embryo induction, embryonic maturation and germination, and plant regeneration through the induction of embryogenic callus from spliced embryos of the elm.

An object of the present invention is to provide an in-flight breeding method of elm tree through somatic embryogenesis that induces embryogenic cells and induces somatic embryos based on mature seed embryos of elm tree.

The present invention for achieving the above object is a step of inducing somatic embryogenic tissue by culturing the zygote embryo of the surface sterilized Chunjam; Culturing the induced somatic embryogenic tissue to maintain and propagate the embryogenic tissue; Inducing somatic embryos by culturing embryogenic tissue; Inducing a plant by culturing somatic embryos; It provides an in-flight propagation method of the elm tree, which comprises the steps of purifying the plant by transplanting the plant into artificial soil.

Hereinafter, the present invention will be described in more detail.

In-flight propagation method of the elm tree according to the present invention comprises the steps of inducing somatic embryogenic tissue by culturing the zygote embryo of the surface sterilized seed elm; Culturing the induced somatic embryogenic tissue to maintain and propagate the embryogenic tissue; Inducing somatic embryos by culturing embryogenic tissue; Inducing a plant by culturing somatic embryos; Transplanting the plant into artificial soil consists of the steps of purifying the plant.

In the above method, surface sterilization was performed after washing the seeds of the young mulberry tree, from which pulp was removed, with water added with Tween 20, 1) immersing in 65-75% ethanol for 3-5 minutes, and 2) adding Triton X-100. Immerse in 1.5 ~ 2.5% (w / v) sodium hypochlorite (NaClO) solution for 30 ~ 40 minutes and sterilize 3 ~ 20% by dipping in 0.1 ~ 0.2% (w / v) Honghong (HgCl ₂ ) solution It is preferable to sterilize for 30 minutes and wash 4 to 5 times with sterile distilled water.

In the above Triton X-100 is preferably added in an amount of 0.01 ~ 0.05% to 1.5 ~ 2.5% (w / v) sodium hypochlorite (NaClO) solution.

In particular, in order to increase the sterilization effect in the sterilization process 2) and 3) by using an alcohol lamp to shake to about 50 ℃ warm state, it is preferable to perform surface sterilization.

Induction of somatic embryogenesis tissues was determined by the condition of cancer culture in MS medium containing 1.0-2.0 mg / L 2,4-D, 2-5% sucrose and 0.2-0.5% gelite. It is preferable to incubate for 4 to 8 weeks.

The maintenance and proliferation of the embryogenic cells induced above were performed by culturing the somatic embryogenic tissue in MS medium containing 1.0-2.0 mg / L 2,4-D, 2-5% sucrose and 0.2-0.5% gelite. It is preferable to incubate at three weeks of subculture under the conditions of.

The somatic embryogenic tissue was cultured in a 1 / 2MS medium containing 0.01-0.05% activated carbon and 0.05-0.5 mg / L absicic acid torpedo stage embryo or early cotyledonary stage. Induce somatic embryos of embryos.

Induce the plant by culturing the induced somatic embryos in MS medium containing 1.0 ~ 10.0mg / L gibberellic acid.

Purification of the above-derived plant is preferably transplanted in artificial clay and then purified in a greenhouse where high humidity of 90 to 95% is maintained.

The present invention is to investigate the conditions of the appropriate growth regulators on ① induction of embryogenic cells from mature seed embryos of young radish, radish, to study the efficient in-flight propagation method of Brussels elvis. The purpose of this study was to investigate the effects of ABA, to investigate the effect of gibberellic acid (GA ₃ ) on the maturation and germination of somatic embryos, and to examine the soil and environmental conditions for soil purification of regenerated young plants.

The present invention is to investigate the effect of growth regulators on callus induction and embryogenic callus induction of seeds collected from Seorak and Bangtaesan, as well as Ebcitic acid (ABA, absicsic acid) on somatic embryo induction, maturation and germination of somatic embryos And gibblelic acid (GA ₃ ) were investigated and a series of methods for plant formation and soil transplantation were developed. In particular, by developing a method to induce somatic embryos by continuously proliferating embryonic cell line (cell line) to be able to form plants whenever necessary. According to the present invention, it is expected that large-scale breeding of the elm tree, which has not yet been established for efficient breeding, can be used as a resource plant and restore natural habitats of destroyed natural vegetation.

Hereinafter, the specific configuration and operation of the present invention have been described through specific experiments and examples, but the scope of the right of plant formation technology through induction of embryogenic cells from zygomatic embryos and other tissues of the elder tree similar to the disclosed material of the present invention is as follows. It is not limited to the embodiment described in the.

Example

Example 1 Harvesting of Seeds

In the present invention, the splicer embryos of mature seeds were used as sections, and the material was taken from Seorak and Bangtaesan in August 2003. The seedling tree is distributed only in high mountains, so the seed yield rate is low and the seed germination rate is very low. Seed varieties were very difficult, and dozens of seeds were collected by dividing the mountainous area without any individual species. Seeds were transported to the laboratory the next day and refrigerated at 4 ° C until use.

Example 2. Surface Sterilization for Culture

Unlike common plant materials, the seeds of the echidna are thickly enclosed by internal and external epithelium, and the surface of the epidermis is covered with numerous small thorns, making surface disinfection very difficult. In the present invention, the seeds were placed in a Erlenmeyer flask and completely washed with tap water containing 0.03% of Tween 20, and then sterilized in the aseptic phase as follows.

1) Soak in 70% ethanol for 3 minutes, 2) transfer to 2% NaClO solution with 0.03% Triton X-100, sterilize for 30 minutes, and 3) finally 0.2% (w / v) HgCl ₂ ) solution was sterilized for 20 minutes and washed four times with sterile distilled water.

In the above sterilization process 2) and 3), the surface sterilization was performed by shaking with warming to 50 ℃ using alcohol lamp to increase sterilization effect. After surface sterilization, the seeds were immersed in sterile distilled water for 30 minutes or more, and only the healthy seeds were selected under a microscope, the seeds were cut with a sharp dissection knife, and the spliced embryos were taken out and placed in a prepared medium.

Example 3. Media Composition and Preparation for Embryonic Tissue Induction

As a medium for inducing embryogenic tissue from mature embryos, MS medium (Murashige and Skoog, 1962), which is generally used in plant tissue culture, was used as a carbon source, 3% sucrose and 0.3% gelite as a hardener. (gelrite) was used by processing. Here, 1,000 mg / L L-glutamine (Sigma) was treated with amino acids, and 1.0 mg / L or 2.0 mg / L 2,4-D (2,4-dichlorophenoxyacetic acid) alone or 2, was used as a plant growth regulator. 0.01 mg / L TDZ was mixed and treated with 4-D, respectively. Acidity was adjusted to 5.8. L-glutamine was added separately by filtering when the temperature of the medium cooled to about 50 ° C. after the heat disinfection of the medium, and the aliquots of the medium were added in a plastic Petri dish (green cross) of 87 × 15 mm size. Dissolve and solidify overnight at room temperature before use.

Example 4. Induction and Proliferation of Embryonic Tissues

After 4 weeks of culture, the sections were passaged in hormonal-free medium supplemented with 20 g / L sucrose and 0.3% gelite in a 1 / 2MS medium in which the amount of salt was cut in half to induce embryonic tissues by cancer culture. After 8 weeks of culture, white fragile embryogenic callus was induced. Embryonic callus was treated with 1,000 mg / L L-glutamine, 1.0 mg / L 2,4-D, 5% sucrose, 0.5% gelite. Embryonic tissues were proliferated by passage in MS medium at intervals of three weeks.

Example 5. Induction of Somatic Embryos

Absuccinic acid (ABA) in medium supplemented with 20 g / L sucrose, 0.02% activated charcoal, and 0.3% gelite in a 1/2 MS medium in which the amount of salt was cut in half to induce somatic embryos from embryonic tissues. Was treated with 5 concentrations (0.00, 0.05, 0.10, 0.20 and 0.50 mg / L), and about 0.5 g of embryogenic callus was transplanted into each disposable shale, and 4 weeks after the culture, the torpedo-to-initial cotyledon was induced. Somatic embryos were examined. The culture was incubated in a culture chamber at a temperature of 24 ° C. under illumination (40 μmol m ⁻² s ⁻¹ ) for 16 hours per day.

Example 6. Germination and Plant Formation of Somatic Embryos

In Example 5, embryos grown to the initial cotyledon type were selected and treated with 3% sucrose and 0.3% gelite in MS medium treated with gibberellic acid (GA ₃ ) to test germination and plant formation. Gibberellic acid (GA ₃ ) was added to the medium by filtering, and re-differentiated young plants were passaged in 1 / 2MS medium (10 × 5 cm culture bottle, 30 mL medium) to promote growth.

Example 7 Soil Transplantation and Purification

In Example 6, young plants germinated from somatic embryos (about 7 to 10 cm) were removed from the culture vessel and carefully washed with gelled water from the roots and transplanted into artificial clay. Topsoil was used by mixing PKS2 and perlite in an equivalent volume ratio. After transplantation, water was sufficiently irrigated and maintained at high humidity (over 90%) by periodic watering in the greenhouse. Two weeks after transplantation, the humidity was gradually reduced to adapt to the external environment.

Effect of Medium and Growth Regulators on Callus and Embryonic Tissue Induction from Seeds and Leaves Medium, mg / L  Callus induction rate (%)  Embryonic tissue induction rate (%) leaf strain leaf strain MS + 2,4-D 1.0 100 71.0 0 1.2 MS + 2,4-D 2.0 100 72.0 0 0.8 MS + 2,4-D 1.0, TDZ 0.01 100 36.0 0 0 MS + 2,4-D 2.0, TDZ 0.01 100 36.0 0 0

Effect of Ebcitic Acid (ABA) on Somatic Embryogenesis in Embryonic Tissues Medium, mg / L Torpedo boat / plate Early cotyledon 1 / 2MS control 20 < 2.0 ± 0.5 1 / 2MS + ABA 0.05 100 < 3.3 ± 0.9      + ABA 0.1 100 < 6.8 ± 2.4      + ABA 0.2 100 < 1.8 ± 1.2      + ABA 0.5 100 < 0.3 ± 0.02

* All mediums are treated with 0.02% activated carbon

Effect of Gibberellic Acid on Germination and Regeneration of Somatic Embryos  Medium, mg / L Cultured somatic embryos Germinated somatic embryo frequency (%) % Of reprogrammed somatic embryos  MS control 130 106 (81.5) 13 (10.0) MS + GA ₃ 1.0 130 126 (96.9) 32 (24.6) + GA ₃ 3.0 123 116 (94.3) 33 (26.8) + GA ₃ 5.0 133 131 (98.3) 47 (35.3) + GA ₃ 10.0 122 109 (89.3) 18 (14.3)

As described above, the present invention induces embryogenic callus from the zygote embryo, maintains and propagates the embryogenic callus, and induces the maturation and germination after seed germination of the elm elm, which has low seed deletion and low fertility. The process can lead to a complete plant.

Based on these results, it is very promising as a medicinal species, but it is expected to produce seedlings through in-flight mass production of rarely endangered species due to overfishing, and to restore the habitats using the same, and to preserve germ quality through cryogenic storage of embryogenic tissues. Therefore, it is expected that genetic resources that will be extinguished will be preserved off-site. In addition, the data of the present invention may be usefully utilized as an in-flight breeding technique of similar rare endangered species, and may be expected to be applicable to the application of advanced biotechnology such as gene insertion using embryogenic cell lines.

<References>

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Claims (7)

Inducing somatic embryogenic tissue by culturing the conjugated embryos of the surface sterilized seedlings; Culturing the induced somatic embryogenic tissue to maintain and propagate the embryogenic tissue; Inducing somatic embryos by culturing embryogenic tissue; Inducing a plant by culturing somatic embryos; Implanting the plant in artificial clay to purify the plant; The surface sterilization was carried out by washing the seeds of the mulberry tree from which pulp was removed with water added with Tween 20, immersing in ethanol, immersing in sodium hypochlorite (NaClO) solution containing Triton X-100, and then dissipating (HgCl ₂ ) The sterilization by sterilization by dipping in a solution, the in-flight propagation method of Sesame elm. delete The method according to claim 1, wherein the induction of somatic embryogenic tissue is performed by zygote embryos of seedlings from 1.0 to 2.0 mg / L 2,4-D, MS containing 2-5% sucrose and 0.2-0.5% gelite. In-flight propagation method of persimmon elm, characterized in that cultured for 4 to 8 weeks under the conditions of cancer culture as a medium. The method according to claim 1, wherein the maintenance and proliferation of embryogenic cells is performed in MS medium containing somatic embryogenic tissue in 1.0-2.0 mg / L 2,4-D, 2-5% sucrose, 0.2-0.5% gelite. In-flight propagation method of elm tree, characterized in that the subculture every two to three weeks under the conditions of cancer culture. The method of claim 1, wherein the induction of somatic embryos is characterized in that the embryogenic tissue is cultured in 1 / 2MS medium containing 0.01-0.05% activated carbon and 0.05-0.5 mg / L absicic aci. Propagation method of zelkova tree to make. The method of claim 1, wherein the induction of a plant is a somatic embryo cultured in infuser of MS elm, characterized in that the culture in MS medium containing 1.0 ~ 10.0mg / L gibberellic acid. The method of claim 1, wherein the purification of the plant is transplanted to artificial clay, and then purified in a greenhouse where high humidity is maintained at 90% or higher.

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