KR100257991B1 - Propagation of eleutherococcus senticosus max. by embryo - Google Patents

Abstract

PURPOSE: A method for propagating eleutherococcus senticosus max is provided to abandon the embryos by cultivating the root tissue of the eleutherococcus senticosus max to specialize the plants. CONSTITUTION: For propagating eleutherococcus senticosus max by using embryos, the little root tissue of the eleutherococcus senticosus max is cultivated in culture fluid containing 2,4-D(plant growth control material) of 0.01 to 4mg/l to abandon the embryos. Then, the embryos callus taken from the embryos is cultivated in an MS culture fluid having a quarter salinity of a basic MS culture fluid. In this way, the embryos are largely propagated. After that, the embryos are unified by using ABA(Abscisic acid) culture fluid of 0.1 to 1.0mg/l. The eleutherococcus senticosus max is specialized in polyamines of 5 to 20mg/l containing spermine or spermidine instead of the plant growth material. Finally, the specialized eleutherococcus senticosus max is transplanted in soil.

Description

Breeding Method of Prickly Pear Using Somatic Embryos

The present invention relates to a method of culturing and growing a tissue of prickly eagle.

More specifically, the present invention relates to a propagation method of culturing root tissues of prickly gallium in solid medium and liquid medium to induce somatic embryos and to multiply them, and to re-differentiate plants and transplant them into soil. In spite of its effectiveness, it can be widely used to produce large quantities of prickly pears without seed in Korea.

Eleutherococcus senticosus Max. Is a deciduous shrub belonging to the genus Ogalpiaceae, which is known to grow natively in southeastern Siberia, parts of Hokkaido, parts of China, and northern and Deogyusan areas of Gangwon-do, Korea. Prickly Pear is a useful medicinal plant called Siberian ginseng, which can be used as a medicinal herb or muscle with various effects such as tonic, neuralgia, hypoglycemic, antistress, stamina reinforcement, and stroke.

Gaiogalpi has a feature that is distributed more like a thin hairs on the stem than other galgalpi. This production is small enough to be hard to find even in the Kyungdong market, where various herbal medicines are traded. Most of the raw materials for prickly pears depend on imports, except that they are currently cultivated on a small scale. In fact, some pharmaceutical companies (Gangnam Pharmaceuticals, Dongshin Pharmaceuticals) import them and manufacture and sell psorigal extracts under the trade names Elle-F and Elcok.

These thorns are difficult to produce in large quantities because the breeding technology using seeds is not made because the seeds are not deleted in the climate and soil of Korea. In particular, thorny gogalpi is very expensive in Korea, and is selling more than 100,000 won (Kim Jae-gi, 243-0618) for water duck cultivation in Chunseong-gun, Gangwon-do. Therefore, if you develop a method or technology for mass production of seedlings, you can earn a high farm income through its cultivation and reduce the foreign currency required to import them.

So far, various attempts have been made to breed seeds because the seeds are not deleted in Korea. Specific examples of successful breeding techniques have been successful germination of seeds introduced from abroad through the process of ripening and dormant breakthrough (Pak, Moon-soo, 1994). 35 (3): 86-91) In addition, there was an example of cutting the stem part of prickly thorn and cutting it into sand or soil, but the cutting rate was less than 30% and breeding rate was very low.

In addition, as a result of differentiation of callus and plants by incubating seedings of immature embryos with foreign embryos, which have been ripened in Murashige and Skoog (MS) medium, (Kim Tae-soo, Ho-ki Park, Park Soo-soo) , Jang Young-sun, Kim Sun-jong, Kim Jong-ho, Seong-gi-gi, 1994, Studies on Reproduction of Prickly Pear III, Callus Organic and Plant Regeneration by Immature Embryo Culture, Journal of the Korean Society of Breeding, 26: 134-1135). However, few studies have been conducted on tissue culture for breeding prickly pears in Korea and abroad, and research on the reproduction (cutting and tissue culture) of prickly pears is being conducted in research institutes, universities, and farms. No results are obtained.

Accordingly, the present inventors have conducted research for breeding prickly falcons, which are difficult to obtain seeds, and have found that the somatic embryo can be used to breed them in large quantities, thereby completing the present invention.

An object of the present invention is to provide a method of breeding prickly tortoises by culturing the root tissues of prickly tortoises to induce somatic embryos, and re-differentiating the plant from them.

1 shows the shape of the prickly thorn tree,

Figure 2 shows the somatic embryo formed from the root tissues of prickly gallium by the method of the present invention,

Figure 3 shows the somatic embryo formed by the suspension culture method of the present invention,

4 is a diagram showing a plant grown by suspending the somatic embryo of the present invention in a medium containing a polyamine,

5 shows a regenerated plant obtained by culturing the somatic embryo of the present invention,

Figure 6 shows the plants survived after transplanting the re-differentiated plants to the soil by treating the soil purification process of the present invention.

In order to achieve the above object, the present invention is to culture the young root tissue of prickly gallium in a solid medium or a liquid medium to which plant growth regulators are added, so that the somatic blastocyst is organic, and callus is taken from the somatic embryo to obtain somatic embryos. After mass propagation, the stages of somatic embryos are unified, and the plants are re-differentiated using them, followed by soil purification, and then transplanted into the soil to breed large quantities of prickly pears.

Hereinafter, the present invention will be described in detail.

The present invention obtains its somatic tissues (including stem, leaves, roots) from thorny golgalpi distributed in Korea and cultured it to mass breed thorny golgolpi difficult to obtain seeds in Korea climate and soil, which is the existing As in his study, US seed from foreign countries is used to distinguish tissue culture or organic methods.

Somatic embryos are embryos produced from plant somatic tissues that can become plants when cultured, and only young root tissues form various somatic embryos in various parts of the spiny gall.

First, in order to obtain somatic embryos, the young root tissues of prickly gallium are cultured in Murashige & Skoog (MS) medium containing plant growth regulators, etc. to induce plant somatic embryos (see Table 1). When plant growth regulators 2,4-D (2,4-dichlorophenoxyacetic acid) is preferably used, the concentration is preferably 0.01-4mg / l.

In addition, in order to obtain a large amount of somatic embryos, somatic embryos are taken from the double somatic embryos are grown in large quantities. At this time, the use of MS medium in which the salt concentration is reduced to 1/4 in the composition of the MS medium shown in Table 1 can more effectively propagate somatic embryos (see FIG. 3).

In addition, in order to use the somatic embryo as an artificial seed, somatic embryos present in various stages such as globular, heart, torpedo, cotyledon, clump, etc. are grown. The regulator is added to homogenize in uniform steps.

The growth regulator used at this time includes ABA (abscisic acid), the ABA concentration is preferably 0.1-1.0mg / ml. In addition, in order to uniformize the stage of somatic embryos, the sugar concentration may be increased (Komatsuda et al., 1992: plant cell, tissue and organ culture 28: 103-113), or ammonium nitrate (3, 4-15 mM) (Tremblay L. and Tremblay FM, 1991, Plant science, 79: 233-242), or using a medium with low or no auxin concentration.

Further, in order to differentiate the somatic embryos obtained from the suspension culture into complete plants, the somatic embryos are transferred to a solid medium or a liquid medium to which no growth regulator is added. At this time, if polyamine is added to the medium necessary for the differentiation of the plant, the rate at which the somatic embryo re-differentiates into the plant increases (see FIG. 4). In this case, spermine, spermidine, or the like may be used as the polyamine. When the somatic embryo obtained above is transplanted using MS medium or the like, the plant is re-differentiated and organically formed into a complete plant (see FIG. 5).

In addition, in order to transplant the re-differentiated plants in the soil, using a variety of soils to go through the purification process and to examine the survival rate of the thorns. At this time, the purification process is carried out under light conditions, it is preferable to perform about 10-20 days at 20-25 ℃ temperature. In addition, the use of vermiculite and vermiculite: perlite soils as soils is suitable for organic young seedlings.

Hereinafter, the present invention will be described in detail with reference to Examples.

The following examples illustrate the invention and are not intended to limit the invention.

Example 1

[Organization of Somatic Embryos from Root Tissue]

Tissue culture materials were prepared using the leaves, young roots and stem tissues of P. falciparum and the concentrations of plant growth regulators 2,4-D, IAA (indole-3-acetic acid), TDZ (Thidiazuron), and kinetin. Different MS solid medium was wounded. 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 (agar) was added 0.8%. Each medium was dispensed into test tubes by 10 ml, which was autoclaved for 15 minutes under conditions of 121 ° C. and 1.5 atm and solidified with a solid medium.

In order to form somatic embryo callus, the leaves, roots and stem tissues of Korean thorns grown in greenhouses are used. Materials are taken from the greenhouse, washed twice with distilled water, and soaked in 70% ethanol for 30 seconds and then sterilized. Washed with water and aseptically treated with NaOCl 0.3-0.5% solution for 10 minutes. The treated material was again washed 5 times in sterile water, cut into 5mm x 5mm size and was wound on the medium used in each experiment. When the leaf and stem tissues were cultured, the somatic embryonic callus was not formed at all and the non- somatic embryonic callus was intermittently formed. When root tissues were cultured, somatic embryo callus was formed in 2,4-D 4 mg / l medium and the formation rate was 50%. Therefore, in the present invention, it was confirmed that root tissue is suitable for somatic embryogenesis and that 2,4-D should be added to the medium (see FIG. 2).

Example 2

[Proliferation of Somatic Embryonic Callus]

In order to propagate somatic embryo callus of P. aeruginosa in Example 1, MS medium completely dissolved 3% sugar by varying salt strength, and then adjusted the pH to 5.7 and agar. It was prepared by adding 0.8%. This was autoclaved for 15 minutes under conditions of 121 ° C. and 1.5 atm or higher, and each medium was dispensed into 30 ml of Petri dishes to coagulate with a solid medium. After 60 days of culture, the somatic embryo callus and the somatic embryo were propagated as shown in FIG.

As a result, the formation rate of somatic embryos and the growth rate of somatic embryo callus were the best in 1/4 MS medium having low salt strength. In MS medium based on salt concentration, torpedo embryo formation and plant regeneration were high. Therefore, it was found that it was effective to use 1/4 MS medium for propagating somatic embryos of prickly gallium, and MS medium for plant differentiation.

Example 3

[Generation and Proliferation of Somatic Embryos]

In order to generate and propagate somatic embryos using proliferated somatic embryos, 3% sugar (sucrose) was completely dissolved based on MS medium, and then treated with plant growth regulators. After the growth regulator was added, the pH was adjusted to 5.7, and 0.8% of agar was added. This was autoclaved for 15 minutes under conditions of 121 ° C. and 1.5 atm and each medium was dispensed into 3 ml petri dishes and solidified with a solid medium. The results of incubation in MS medium with different composition of growth regulators are shown in Table 4.

As a result, the plant regeneration was not performed at all after 2,4-D, which is a growth regulator, while somatic cell formation was very good when 2,4-D was treated at 0.1, 1 mg / l. In addition, when BA (benzyl adenine) was treated with low concentrations of 0.01 mg / l BA, somatic embryogenesis and plant regeneration were simultaneously observed. In addition, TDZ treatment resulted in regeneration of plants, and treatment of TDZ with high concentrations of 1 mg / l resulted in 100% plant regeneration (see Figure 3).

Example 4

[Proliferation of Somatic Embryonic Callus through Suspension Culture]

Somatic cells formed in the solid medium were suspended in culture using liquid medium. In order to propagate somatic embryo callus by suspension culture, 3% sucrose was completely dissolved based on MS medium, and then treated with plant growth regulators. At this time, the growth regulator was added, and then the pH was adjusted to 5.7. Each medium was dispensed into 50 ml of 250 ml flasks, which were autoclaved at 121 ° C. and 1.5 atm for 15 minutes. The results of culturing in liquid MS medium by varying the type and composition of growth regulators are shown in Table 5.

As a result, overall somatic embryos were better formed in the 2,4-D treated group than IAA as a growth regulator, and 507 somatic embryos were formed at the 2,4-D concentration of 1 mg / l. In addition, somatic embryos were formed step by step, and the most torpedo-shaped embryos were formed at 186 to 2,4-D concentration. When IAA was treated at 1 mg / l concentration, the number of somatic cells formed was 171, but when 2,4-D was treated at 1 mg / l concentration, 507 somatic embryos were formed. Therefore, it was found that 3 times more somatic embryos were formed in 2,4-added medium, and 2,4-D concentrations of 1 mg / l and 2 mg / l were suitable for forming somatic embryos in liquid medium. These results are the basic and actual data for the experiments that are useful when using large liquid containers such as 500ml, 1l, and 2l to form somatic embryos, and when using large-capacity containers such as bioreactors. Will be.

Example 5

Plant Regeneration by Polyamine Treatment

To obtain the plant from the somatic cell culture formed in the suspension culture, MS solid medium or basic liquid medium without growth regulator was added. In this example, 3% sugar based on MS medium is used to differentiate the somatic cell culture. (Sucrose) was completely dissolved and then treated with different concentrations of spermidine and spermidine. At this time, by adjusting the pH to 5.7, each medium was dispensed into 50 ml 250 ml flask, which was used by autoclaving for 15 minutes under conditions of 121 ℃, 1.5 atm or more. The results of incubating the somatic embryos in the liquid medium with different compositions of spermine and spermidine as polyamines in the basal medium and the basal medium are shown in Table 5.

As a result, 359 plants were re-differentiated in a 250 ml flask containing 50 ml basic liquid MS medium without polyamine treatment, but more than 1100 plants were split in a flask treated with 5 mg / l spermine or 5 mg / l spermidine, which used only the basic liquid medium. At least four times more plants were re-differentiated. Treatment with spermine or spermidine 10 mg, 20 mg also re-differentiated more than 900 plants, 2.7 times more plants than did not. Therefore, the present invention is the first report in Korea and abroad as a groundbreaking result of the distribution of somatic embryos of prickly gallium obtained from suspension culture into four or more times the plants (see FIG. 4).

Example 6

[Unification of Somatic Embryos]

In order to make artificial seeds from somatic embryos formed by suspension culture, the somatic cells of various stages such as globular, heart and torpedo should be unified into uniform somatic cells. Therefore, 3% of sugar (sucrose) was completely dissolved based on MS medium in this example, and then treated with different ABA concentrations. At this time, the medium was adjusted to pH 5.7 and each medium was dispensed into 50 ml 250 ml flasks, which were autoclaved at 121 ° C. and 1.5 atm for 15 minutes. The result of culturing somatic embryos in liquid MS medium with different ABA compositions is shown in Table 7.

As a result, when ABA was treated at a low concentration of 0.01 mg / l, almost all somatic embryos differentiated into plants and remained low in somatic embryos, but ABA was increased to 0.01, 0.1, 0.5 mg / l. Almost all somatic embryos were homogenized to torpedo-shaped torpedo embryos. Therefore, it was confirmed that it is most suitable to treat ABA at a concentration of 0.5 mg / l to unify somatic embryos. These results will be invaluable in the process of homogenizing somatic embryos.

Example 7

Plant Regeneration by Soil Purification Treatment

After transplanting the regenerated plants grown through the somatic cell culture into the soil, the plants were washed to examine their viability, and then the soil, hultan, vermiculite, perlite, vermiculite and perlite were 1: 1. Transplanted into mixed soil. After transplantation, the cells were purified under 23 ℃ and 16 hours light condition. After 20 days of acclimation, they were transferred to a greenhouse and their surviving plants were examined.

As a result, 82.1% of the re-differentiated plants were purified to produce young seedlings in the vermiculite: pearlite 1: 1 mixed soil, and 78.6% of the re-divided plants survived in the vermiculite soil. In addition, although the survival rate of 50.0% was observed in the soil of perlite, the seedlings were formed better than the soils of top soil and Huntan. Therefore, it was confirmed that the plants re-differentiated in the cabin can be maintained as young seedlings through the soil purification process.

In the present invention, by culturing the cultivated prickly pears, the somatic embryo is regenerated and the plant is re-divided from the plant to effectively transplant into the soil, so that seed is not formed and the seeding is not well-produced. have. Specifically, the method of the present invention is a breakthrough method suitable for rapidly breeding a large number of thorns in a short time since one can obtain more than 1000 plants in one 250 ml flask in 6 weeks.

Claims (2)

Cultivating somatic embryos by culturing the young root tissues of P. falciparum in a solid or liquid medium containing 2,4-D, a plant growth regulator, in a concentration range of 0.01-4 mg / l; 2) taking a somatic embryo callus out of somatic embryos to mass propagate somatic embryos in MS medium whose salt concentration is 1/4 of the base MS medium; 3) unifying the step of somatic embryos using a medium containing ABA (Abscisic acid) as a growth regulator in the concentration range of 0.1-1.0 mg / l; 4) regenerating the plant in a medium containing 5 to 20 mg / l of polyamine containing no plant growth material and comprising spermine or spermidine; And, 5) purifying replanted plants into soil and transplanting them. The method of claim 1, wherein the soil purifying process at 20 to 25 ℃ under the light conditions, 10 to 20 hours of treatment for transplanting the prickly toes characterized in that transplanted to the soil.

Images