KR19980020958A - In-plant seedling production method through single cultivation - Google Patents

Abstract

This invention is intended to facilitate the cultivation of ginseng by producing a large amount of ginseng seedlings in the cabin. Ginseng seedlings are produced by inducing single embryos from mature ginseng cotyledons in murascige and squeegee medium without plant growth regulators and germinating them by low temperature (about 1 ℃) treatment or gibberellin treatment. To promote root and stem development, single germinated embryos are cultured in murasage-squeeged medium without ammonium. According to the present invention is eliminated the hassle of breeding by seeding the seed is easy to grow ginseng.

Description

[Name of invention]

In-plant seedling production method through single culture of ginseng cotyledons

Detailed description of the invention

[Purpose of invention]

The present invention relates to a method of producing in vitro cultured ginseng seedlings by culturing mature ginseng cotyledons in a culture medium of murascige and a scoop without adding a plant growth regulator. It is directed to a method of producing ginseng seedlings by inducing a single embryo directly from single cells of cotyledon epidermis in the basic medium to germinate and grow, and then promote the growth of stems and roots in a medium containing ammonium.

[Technical Field to which the Invention belongs and Prior Art in the Field]

Ginseng is a perennial plant that grows as seeds. When harvesting, the zygotic germ (seed) of ginseng seeds is very immature, and it is necessary to develop immature pears as mature embryos after alluvial treatment for several months before sowing into soil. It must go through a process and at least three years have elapsed before obtaining the next generation of seeds. As such, ginseng takes a long time to germinate seeds of the next generation, and the amount of single harvest is much smaller than that of other plants. Methods of growing ginseng plants have been actively studied.

For example, the root section of ginseng was cultured to obtain callus, and the callus was cultured in-flight to generate somatic embryos, buds, and buds, but the roots did not grow normally. et al., Bot. Zh., 7, 906, 1968; Chang Hising, Theor. Appl. Genet., 57, 133, 1980; Choi et. al., Kor. J. Ginseng Sci., 5, 35, In addition, studies were conducted to induce somatic embryos by culturing cotyledon tissues of immature ginseng and to induce ground and roots from these somatic embryos, but no ginseng plants with normal stems and roots were obtained.

As mentioned above, various researches have been conducted to obtain in-flight seedlings of ginseng through tissue culture from the roots and cotyledons of ginseng, but until now, it has failed to obtain normal seedlings with well-developed stems and roots. Relies on seeds directly harvested from ginseng.

[Technical problem to be achieved]

The present inventors have continued to study the growth of ginseng, while using the cotyledon of cotyledon as a culture material and cultivating the ginseng cotyledon in the culture medium of Murashige and Scuba without added plant growth regulator, single embryo with normal stem and beak Knowing that this occurred, the present invention was completed.

[Configuration and Function of Invention]

The present invention is directed to the production of ginseng seedlings by direct induction, germination and growth of single embryos from single cells of cotyledon epidermis in basal medium to which no growth regulator is added.

The object of the present invention described above is to use the cotyledon at a specific maturity period as a culture material by utilizing the internal self-development ability of ginseng cotyledons, unlike the conventional method of supplying plant hormones from outside to generate embryos. It is achieved by the in-plant seedling production method of the invention.

According to the present invention, the problem that the roots and / or stems are poorly solved in the conventional in-flight cultured ginseng plant production method is solved, so that the ginseng seedlings can be easily developed and transplanted directly into the soil.

The present invention is to cultivate the mature ginseng cotyledons in the basal culture medium and Muragge without added plant growth regulator, but using the cotyledons of zygote as a culture material to generate a single embryo with normal stem and root, these single batches Germination by low temperature treatment or gibberellin treatment, and then transferred to the musiage and squeegee medium from which ammonium is removed.

In-plant plants produced by the present invention were grown as normal plants when transplanted into the soil.

Hereinafter, the present invention will be described in detail.

Ginseng cotyledons are sterilely cultured in basal culture medium and muskage without growth regulators. In order to determine the rate of embryo development and the rate of development of multiple and single embryos according to the maturation of cotyledons, immature embryos, mature embryos just before germination, seedlings and callus were cultured in the same medium. Experimental results show that not only the single embryos and the embryos are fused with each other, but also somatic polyembroys (hereinafter referred to as multiples), which are fused with parental explants. It turned out that the frequency is different. Experimental results for embryo development frequency are shown in Table 1.

As can be seen in Table 1 above, when the cotyledons of immature embryos were cultured, many times more than single embryos were produced. In embryos immediately before germination, the incidence rate of embryos was lower than that of immature embryos, but the incidence of single doubling than multiple embryos. In the high seedlings, embryos did not occur, and in case of callus, embryos were found to be less in immature embryos or embryos immediately before germination, and multiple embryos were higher than single embryos. In other words, the incidence of single embryos was higher as the cotyledons matured and differentiated.

When the cotyledons were cultured with the cotyledons of the cotyledons in contact with the medium, the rate of embryonic development was reduced by twice as much as when the cotyledons of the cotyledons were in contact with the medium. It is preferable to culture.

In particular, when cotyledon slices were cultured on a culture medium containing only 5% of sugar and 0.7% of agar without adding plant bioregulator to murasage-squeg base salt, growth control agent was produced directly without callus induction in cotyledon base. Induction of the callus by the addition of the first and then induction from the pear is reduced by more than two times compared with the conventional method. When cultured as described above, it takes about two months for the somatic embryos to fully mature until the cotyledon stage. However, after fully matured until the cotyledon stage, they dormant for at least six months and no longer grow. As described above, the single pears that are generated are dormant after growth and dormant after growth, which is a unique feature of ginseng pears.The characteristics of these ginseng pears, unlike other plants that are a problem of early germination, can be stored for several months. It brings the advantages that

When cultured as described above, somatic embryos mature sufficiently in the cotyledon stage, they are transferred to medium containing 1-100 mg / l gibberillin or incubated at low temperature (about 1 ° C.) for at least 4 weeks to induce germination. .

In order to confirm the formation rate of seedlings of single pears and multiple pears obtained from the cotyledon section, the germination test results of single pears and pears under the same conditions are shown in Table 2.

As can be seen in Table 1 and Table 2 above, the somatic embryo suitable for the present invention is a single embryo, and the single embryo has a higher incidence rate as the cotyledon matures and differentiates, and the seedlings having normal stems and roots have a single embryo. It can be seen that it is obtained only at and not at multiple times.

Therefore, in order to obtain a seedling with normal stems and roots, a single embryo should be derived from relatively mature ginseng cotyledon with a high incidence of single embryo and used as a culture material.

In particular, according to the present invention, when the germinated seedlings are grown in the murasage-squeeze medium without ammonium, the roots grow faster and the ginseng seedlings are grown so that the roots can be directly transplanted into the soil. .

Plants with these stems and roots intactly developed were implanted into artificial soils (Perlite: Bermuculite: Pitmos 3: 1: 1). As a result, 91% of single embryos from cotyledons survived.

The method of the present invention described above can be used in genetic engineering breeding method for producing a transgenic plant by introducing a foreign gene, and can be utilized for the production of ginseng seedlings because there is little possibility of generating a variant in culture.

[Effects of the Invention]

According to the present invention, the ginseng seedlings with well-developed roots and stems can be mass-produced in the aircraft, so that the seeding in breeding ginseng plants does not have to be seeded and multiplied, so that the cultivation of ginseng is eliminated and cultivation of ginseng is not required. It becomes easier.

Hereinafter, examples will be described in order to facilitate understanding of the present invention.

EXAMPLE

Example 1

The ginseng unripe seed was alluved in sand at about 10 ° C. for 4-5 months to mature the zygotic embryos to a sufficient size, and after 3 months or more at 4 ° C., the seeds immediately before germination were treated with 70% ethanol. After treating for 1 minute and sterilizing the surface for 20 minutes in 1% sodium hypochlorite solution, washed several times with sterile distilled water and cut cotyledons. The aroma of cut cotyledon slices was added to a Petri dish dispensed with 5 ml of sugar and 0.7% of agar and adjusted to pH 5.8 in 30 ml aliquots of Murashige-Squag, and light-conditioned with 1900 lux white fluorescent lamp for 16 hours. Somatic embryos were generated by culturing for 5 weeks in a culture chamber maintained at 24 ° C.

The somatic embryos were cultured for 60 days, matured sufficiently in the cotyledon stage, and then immersed in Murashige-Squeeze medium containing 5 mg / l gibberellin, and then subjected to bright conditions for 16 hours using a 1900 lux white fluorescent lamp. Incubate in the culture chamber. In this state, the pear becomes green and, after one week of ringing, germinated seedlings are obtained.

Example 2

Somatic embryos matured in the cotyledon stage in the method of Example 1 were left in Daesan cultured in Murashage-Skug medium to which 5 mg / l gibberillin was added, and left for about 4 weeks at 1 ° C., followed by Murashige-Suku at room temperature. Transfer to the medium and incubate.

Example 3

Germinated seedlings obtained in Example 1 were separated using tweezers one by one in order to generate more healthy roots so that the germinated seedlings could be easily adapted to the soil, and then in murasage-squeegous medium having different concentrations of ammonium salts. Transfer to grow. In order to check the growth of root and ground portion in the murasigge-squeg medium with different concentrations of ammonium salt, the nitrogen source (1650 mg / l ammonium nitrate and 1900 me / l potassium nitrate) in the medium was changed and tested as shown in Table 3. The growth length of roots and the incidence of terrestrial outbreaks from single ginseng with roots were measured. Test results are listed in Tables 3 and 4.

According to Table 3, the roots were not developed in the medium in which potassium nitrate was not used, and in the medium in which an excess of ammonium nitrate and potassium nitrate was doubled to the basic concentration.

Root growth was poor in the medium in which ammonium nitrate and potassium nitrate were mixed, and the growth of root in the medium using only potassium nitrate alone.

Therefore, in order to obtain ginseng seedlings with roots developed enough to be directly transplanted into the soil from germinated ginseng seedlings, it was found that the murasique-squeeged medium in which ammonium was completely removed was used.

According to the aforementioned Table 4, among the seedlings with roots, about 48% of single shoots are formed from a single root, and about 35% of multiple shoots are generated from a single root. In about 17% of cases, a total of 83% were found to be normal seedlings.

Based on the results of the above-described examples, the mature ginseng cotyledon immediately before germination was overlaid on a muri-sique-squeg base medium containing no plant hormone and 5% sugar to induce a single pear, For example, when germinated by about 1 ° C. or gibberellin, ginseng seedlings in which roots and stems are normally developed are obtained. The germinated seedlings are transplanted when cultured in a murasage-squeeze medium having a nitrogen source from which ammonium is removed. It can be seen that ginseng seedlings with well developed roots and stems can be obtained.

Claims (7)

A method of producing a seedling plant in ginseng seedlings, in which the ginseng cotyledons are grown from ginseng cotyledons to induce a single embryo and germinate by cultivating mature ginseng cotyledons in murascige and scoop basic medium without plant growth regulators. 2. The method of claim 1, wherein the single germination is in a Murashike-Skug base medium comprising gibbelillin. 3. The method of claim 2, wherein gibberillin is contained 1-100mg / l. The method of claim 1, characterized in that the single germination is made by cold treatment. The method according to claim 4, wherein the low temperature treatment is performed at about 1 ° C. 2. The method of claim 1, wherein the murasigge-scuba badge used to induce a single pear comprises about 5% sugar. The method of claim 1, wherein the germinated ginseng seedlings are cultured so that roots and stems are grown to the extent that they can be transplanted into the soil in the murascige-squag medium without ammonium.