KR20110014255A - Plant stem cell line derived from quiescent center and method for isolating the same - Google Patents

Abstract

PURPOSE: A quiescent center-derived cell line and a method for isolating the same are provided to induce and culture single cell-originated cell line without dedifferentiation. CONSTITUTION: A quiescent center-derived cell line of a rice plant comprises: a step of removing peel of rice plant seeds and sterilizing with 70% ethanol for one minutes; a step of immersing in 2% sodium hypochlorite for one hour and washing with distilled water once or twice; a step of removing moisture completely; a step of seeding the dried seeds in an N6 medium at 25 degree Celsius for five days; and a step of collecting a root having the quiescent center from the rice plant. Other plant to be used for isolating the quiescent center is corn, pea, oat, onion or Arabidopsis thaliana.

Description

Plant Stem Cell Line Derived from Quiescent Center and Method for Isolating the Same}

The present invention relates to a cell line derived from a center of plant lysis and a method for separating the cell line from the center of plant lysis, and more particularly, to a homogeneous cell line derived from a lysis center center derived from a single cell origin, Separation method.

Plants used to mean food resources in the past, but now they are expanding as a source of a wide range of chemicals, including pharmaceuticals, flavorings, pigments, pesticides, and dyes. In particular, since most of the plant-derived useful substances have physiological activities such as antiviral, antibacterial, anticancer, and antioxidant ability, they are attracting attention as an ideal resource for development as a new drug, and the relationship between chemical structure and activity of many plant-derived substances Researches are actively underway to identify the problem.

However, bioactive substances are difficult to develop as pharmaceuticals, and the main reasons thereof are as follows. First, the content of bioactive substances in plants is extremely limited. Second, the growth rate of plants is very slow. Third, plant-derived bioactive substances are present only in small quantities within certain organs of the plant. Fourth, environmental issues such as natural degradation are involved. Fifth, in the case of a plant-derived physiologically active substance, the chemical structure is very complicated, so a multi-stage polymerization process is required, and thus there is an economic problem in which the production cost is very high. Thus, it has been very difficult to commercially and stably supply plant-derived bioactive substances.

However, the plant cell culture method, which is one of biotechnology techniques, has long been evaluated as the most ideal technology capable of stably supplying plant-derived useful materials without causing environmental problems. According to Republic of Korea Patent Publication 1995-0000870 (1995. 01. 03) the production of useful substances by plant cell culture has a number of advantages over the method by direct extraction from plants. In particular, unlike conventional extraction methods, it is possible to continuously produce without being influenced by the external environment, and is considered as the best way to solve the problems such as ecosystem destruction. However, despite much interest and efforts in plant cell culture, only a few examples of successful industrialization are still. This is because variation in cell proliferation and productivity remains a major problem in many plant cell cultures.

In the case of using plant cells in the plant expression system, since the differentiated tissues of the plant cells, such as leaves, stems, seeds, and the like, are permanent tissues that have lost dividing ability, dedifferentiation process is essentially preceded to convert them into cell lines having dividing ability. The dedifferentiation process means dismantling a state in which a tissue or cell has already been differentiated to perform a specific function when cultured using a tissue or organ of a plant. However, during this dedifferentiation process, severe variations in cell lines can occur due to chromosomal variations.

In particular, the production of useful substances through plant cell culture can be industrialized only if the rapid cell proliferation and high metabolite production capacity are maintained stably for a long period of culture, but most cell lines undergo numerous mutations different from the original by passage culture. You will come across. Therefore, in order to overcome such a mutation problem, a method for obtaining a genetically stable cell line in the production of useful substances through plant cell culture was urgently needed.

On the other hand, the surface of the roots is very large because plants have to absorb enough water and inorganic substances for growth. At the end of these roots are myomitotic cells, which divide, expand, elongate, and differentiate to form the primary tissue of the root. The myomitotic tissue is protected by the root canal (root thimble), and the cells in the middle of the myomitotic tissue divide very slowly and are called the division delay center. Around the division, the primary cells that make up the first stage meristem are surrounded. have.

However, despite the importance of plant root system, studies on the physiological characteristics of myomitotic tissues are insufficient. In other words, studies on the physiological characteristics of the delayed division center using plants such as corn or cultivation of them in the culture medium have been developed as roots, but various roots of root canals, vascular tissues, endothelial cells, endothelial cells, fitzels, epidermis, etc. There was no case where only division division center was isolated and established as cell line.

Therefore, since the divisional division center is the most genetically stable cell tissue, it is possible to study the development and genetic origin of the plant by separating it, and thus, the development of a method for separating the homogeneous cell lines derived from the division division center is required. In addition, in recent years, stem cell biology has recently emerged, and many experiments related to stem cells have been conducted, such as studies on signals involved in developmental processes. However, the method of isolating and culturing stem cells in animals has been established early, but there is no study on stem cell separation in plants. The induction and separation of cell lines derived from divisional delay centers are expected to accelerate the development of plant stem cell biology.

Therefore, the present inventors have made effort to develop a plant cell which can be used for a plant expression system without dividing a homogeneous cell line originating from a single-cell origin originating from a mitotic retardation center and a dedifferentiation process. As a result, The inventors of the present invention have found that the cell line has almost no mutation during long-term culture, enables stable culture, and has a high survival rate upon cryopreservation.

It is an object of the present invention to provide a cell line derived from a division delay center capable of stable culture.

It is another object of the present invention to provide a method for isolating a cell line derived from a mitosis-delayed center without a dedifferentiation process.

In order to achieve the above object, the present invention provides a method for separating the delayed division center-derived cell line, characterized in that after culturing the root tissue containing the division delay center of the plant, recovering undifferentiated white tissue.

The present invention also provides a cell line derived from the division delay center of the plant derived from the division delay center of the plant and having the following characteristics:

(a) present as single cell in suspension culture;

(b) exhibits morphological features with larger nuclei compared to cell lines derived from tissues other than the division delay center;

(c) surrounded by mucus;

(d) remain stable without morphological variation in long term culture; And

(e) High survival rate in cryopreservation.

The present invention also provides a method of preserving a plant cell line characterized by freezing the cell line derived from the division-delayed center of the plant.

The present invention has the effect of providing a stable plant cell line and a method for separating the same by inducing and culturing a cell line of unicellular origin without dedifferentiation from the most genetically stable division delay center of the plant constituent tissue.

A homogeneous cell line derived from the cleavage delayed center separated by the method according to the present invention is useful as a research tool for development and genetic origin and is useful for the development of plant stem cell biology. Meanwhile, the cell line derived from the mitotic retardation center according to the present invention is stably maintained without morphological changes for a long period of time, and can be stably and efficiently produced because it is cultured in a single cell state upon suspension culture. Unlike other plant cells, It is possible to construct a plant cell bank using the cryopreservation method with a survival rate of 85% or more. That is, since the cell line according to the present invention is constructed as a plant cell bank, the supply of research materials can be facilitated and the research period using plant cell lines can be shortened.

FIG. 1 (A) is an optical microscope photograph showing the cell line induced by culturing the root tissue containing the cleavage delayed center of rice, followed by separation of the cell line derived from the center of the cleavage delay, and It is the photograph which I cultivated for 4 weeks after it.
FIG. 2 is a graph showing the results of the measurement of root breakage in the root portion of the cleavage delay center, including 2,4-D (A), CPA (B), IAA (C), IBA (D), NAA (E) and picloram And the result is an optical microscope photograph.
FIG. 3 is an optical microscope image of a cell line (a) derived from a root tissue other than the cleavage delayed center of rice and a cell line derived from the cleavage delayed center (b) morphologically.
FIG. 4 is a graph showing the distribution of the proliferation delayed root-containing root tissues, including 2,4-D (A), CPA (B), IAA (C), IBA (D), NAA (E) and picloram And the result is an optical microscope photograph.
Figure 5 shows the morphological variation of root tissue-derived cell lines other than the division delay center according to the culture period.
Figure 6 shows the morphological stabilization of the cell line derived from the mitotic retardation center according to the incubation period.
In FIG. 7, A is an optical microscope photograph (400-fold magnification) of the cell line derived from the divisional delay center, and B is an optical microscope photograph (400-fold magnification) of the root tissue-derived cell line.
Fig. 8 is an optical microscope photograph of a cell line derived from root tissue (a) and a cell line derived from a lysis center (b) other than the lysis center.
FIG. 9 is a graph showing the agglutination rate of the root tissue-derived cell line and the cleavage-delayed center-derived cell line other than the cleavage delay center.
FIG. 10 is an optical microscope photograph showing the survival rates after cryopreservation of the root tissue-derived cell line (a) and the mitotic late-delayed cell line (b) other than the divisional delay center.
FIG. 11 is a graph showing the survival rate after cryopreservation of root tissue-derived cell line and cleavage delayed-center cell line other than the cleavage delay center.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In one aspect, the present invention relates to a method for isolating a delayed division center-derived cell line, characterized in that after culturing the root tissue containing the division delay center of the plant, recovering undifferentiated white tissue.

At this time, preferably, the root tissue containing the division delay center of the plant is obtained by germinating germ-treated plant seeds or using root tissue differentiated from callus derived from a part of the plant. In addition, the medium used in the culture may be any cell line induction medium known to those skilled in the art, but is preferably cultured in any one of N6 medium, MS medium, Gamborg B5 medium, LS medium and KAOM medium. At this time, more preferably, the culture is carried out in a medium containing 2,4-D. Further, in the present invention, the division of the division delay center-derived cell line may be performed after culturing for 3 to 6 weeks.

The "dividing delay center" of the present invention means a hemispherical or discoid single cell group composed of 500 to 1,000 inactive cells in the center of the root apical meristem. These cell populations are known to stay long in the G1 phase and divide in about 15-20 days. They usually remain inactive and re-divide division activity during surgical or radiological injury treatment. That is, when the roots break through the soil, the root canal effectively protects the proximal meristem, but it is not perfect, and the meristem sometimes gets injured while the root is growing. At this time, the cells in the division delay center divide and rebuild damaged meristem and root canal. In addition, the division of the dividing cells is stopped when X-ray exposure, but the division delay center cells are not affected by the X-ray and start to divide immediately to remodel the dividing cells. In other words, the division delay center is a place where genetically stable cells are stored. The cleavage delay center is differentiated into the primary meristems of the roots, the metastatic layer, the basic meristem and the epidermis.

The division delay center can be obtained from the roots of the plant, preferably from the roots of the newsletter germinated aseptically treated seeds, or from root tissues differentiated from callus derived from a part of the plant. At this time, preferably, the root canal is removed from the root end, and a fragment of about 1 mm from the cut surface is obtained and cultured in a cell line induction medium. At this time, prior to the cultivation, the sterilization process can be carried out according to a general method known to those skilled in the art after obtaining the root tissue of the plant, in the case of using the root of the news material germinated aseptically treated seeds, after obtaining the root tissue, It may not be sterilized.

The cell line induction medium may be any known in the art, for example N6 medium (Chu CC, Proc . Symp . Plant Tissue Cult . , Peking, 43, 1978), MS medium (Murashige T. and Skoog F., Physiol Plant, 15:. 473, 1962), Gamborg B5 medium (Gamborg OL et al ., Exp . Cell Res ., 50: 151, 1968), LS medium (Linsmaier EM and Skoog F., Physiol . Plantarum . , 18: 100, 1965), KAO M medium (Kao KN and Michayluk MR, Planta . (Berl.), 126 : 105, 1975), but is not limited to such.

At this time, more preferably, 2,4-D is incubated in auxin. At this time, preferably 2,4-D is included in the concentration of 2mg / L, more preferably in the concentration of 2 ~ 7mg / L. The culture conditions, the culture period, etc. for the specific cell line induction is determined according to the type or characteristics of the plant cells, the practice will be apparent to those skilled in the art.

In the culture, morphological differences between the root tissue-derived cell line and the division-central cell-derived cell line other than the divisional division center are observed. While the heterologous and local differentiation is observed in the root tissue-derived cell line, the division-derived cell line is homogeneous and local. No significant differentiation was observed. In addition, the root cell-derived cell line was yellow when visually observed, whereas the cell division-derived cell line was white and surrounded by mucus. In other words, the cell line derived from the division delay center was "undifferentiated white tissue". The mucus is expected to be musigen, which is secreted by the peripheral cells of the root canal and the epidermal cells of the root and is known as a complex polysaccharide containing sugar, organic acid, vitamins, enzymes, amino acids, and the like.

Thus, based on these morphological differences, only cell lines derived from division delay centers can be selected. Figure 1A is a photograph before separation of the division cell line derived from division delay center, the red circle is a cell line derived from division delay center part, Figure 1B is a photograph obtained after incubation for 4 weeks by separating the induced cell division derived cell line derived after separation.

The recovery of the cell line derived from the mitotic retardation center is preferably carried out after inoculation of the root tissue containing the mitotic retardation center of the plant in the medium, preferably for 3 to 6 weeks, more preferably for 4 to 5 weeks. Approximately 3 to 6 weeks after the inoculation, the cell line derived from the mitotic retardation center is induced, which facilitates the isolation.

Since the division delay center portion is a tissue possessed by all plants, the method of separating a division delay center portion derived cell line of the present invention is applicable to all plants, and thus it is possible to obtain a division line derived from the division delay center from all plants. That is, in one embodiment of the present invention, but the cell line is separated from the division delay center portion of the root tissues of rice and corn, but not limited to this, if the plant having a division delay center portion can apply the method of the present invention belongs to It is obvious to those skilled in the art. For example, rice, corn, peas, oats, onions, Arabidopsis, etc. may be cited as plants capable of obtaining a division-derived center cell line, but are not limited thereto. Examples of physiological characteristics of cleavage delay centers include maize, arabidopsis, and onion ( Allium ). cepa ), oats ( Avena sativa ), and pea ( Pisum sativum ), etc. [ Maize : Georgina Ponce et al ., Plant Cell and Environment, 28: 719, 2005; Keni Jiang et al ., Development , 130: 1429, 2003: Arabidopsis: Noriko Kamiya et al ., The Plant J Ornal , 35: 429, 2003; Peter Doerner, Current Biology , 8: R 42, 1998; Allium cepa : R.Liso, New Phytol ., 110: 469, 1998; Avena sativa : FAL CLOWES , New Phytol ., 129, 1982; Pisum sativum: Peter Doerner, Current Biology , 8: R 42, 1998].

In another aspect, the present invention relates to a cell line of a plant derived from the division delay center of the plant and having the following characteristics:

(a) present as single cell in suspension culture;

(b) exhibits morphological features with larger nuclei compared to cell lines derived from tissues other than the division delay center;

(c) surrounded by mucus;

(d) remain stable without morphological variation in long term culture; And

(e) High survival rate in cryopreservation.

The cell division-derived cell line derived from the present invention exhibits morphological characteristics having a relatively large nucleus, and the size of the nucleus is 2-4 μm, which is larger than that of other cell lines derived from other tissues.

Cell lines derived from the division delay center of the present invention also show very stable proliferation without morphological variation even during long-term culture. In one embodiment of the present invention was found to be cultured stably without morphological changes even when cultured for more than 16 weeks. On the other hand, when prolonged culturing of root cell-derived cell lines, several local differentiations were observed in the cell mass, and especially, the development of the root muscle was remarkably confirmed.

In addition, unlike microbial cells, plant cells are cultured in the form of cell masses rather than single cells. Such cell aggregation causes environmental differences between the inside and the outside of the cell masses, causing mutations in cell proliferation and useful material production. However, the dividing delay center-derived cell line of the present invention is cultured as single cells in suspension culture, so there is no possibility of such mutation.

Thus, the cell line derived from the mitotic delay center according to the present invention can be used in a plant expression system to stably produce a useful substance. Further, the cell line derived from the mitotic retardation center of the present invention can be cultured by a plant suspension culture method, and a specific culture method can be carried out as is known in the art.

In another aspect, the present invention relates to a method for preserving plant cell lines, wherein the plant cell line derived from the division delay center portion of the plant is frozen.

In addition, in the case of plant cells, the survival rate was found to be low during cryopreservation, but the cell division-derived cell line derived from the present invention has a very high cell survival rate of 85% or more when cryopreserved according to a conventional cell cryopreservation method. If the cell line can be cryopreserved, it is possible to construct a stable supply of raw materials and to establish a substantial master cell bank. Thus, a long-term and stable cell line can be supplied using a cell line derived from a division center of division of the present invention.

The world is in the midst of the research material (biological resources) war, and preservation and detoxification of biological resources for the development of various new drugs such as human tissues, plant seeds and microorganisms, cells, genes and so on are emerging as national assets. Therefore, it is necessary to construct a cell line that can collect, preserve and distribute a cell line which is used as an essential data for the research related to life sciences in the age of securing the research material and soon become a national competitiveness. Therefore, it is expected that the supply of research materials will be facilitated in order to build such a plant cell bank, and the research period using plant cell lines will be shortened.

Through the following examples will be described the present invention in more detail. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example  1: rice Fission Delay Center  Isolation of Derived Cell Lines

1-1: Preparation of Plant Materials

The rice seed was removed from the shell, surface sterilized for 1 minute with 70% ethanol in its original state, immersed in 2% sodium hypochlorite solution for 1 hour, and washed once or twice with sterile water. It was washed again with sterile water for 30 minutes and then completely drained.

Dry seeds were grown in N6 medium (CHU MEDIUM, Chu CC, Proc. Symp. Plant Tissue Cultivation, Peking, 43, 1978) and germinated at 25 ° C for 5 days. The composition of the N6 medium is shown in Table 1.

Furtherance density

Macroelements

CaCl ₂ · 2H ₂ O 1.13mM 125.33 mg / L KH ₂ PO ₄ 2.94mM 400.00mg / L KNO ₃ 27.99mM 2830.00 mg / L MgSO _{4 7} H ₂ O 0.75mM 90.27 mg / L (NH ₄ ) ₂ SO ₄ 3.50mM 463.00mg / L Furtherance density

Microelements

FeNaEDTA 0.10mM 36.70mg / l H ₃ BO ₃ 25.88 μM 1.60mg / l KI 4.81 μM 0.80mg / L MnSO ₄ 4H ₂ O 19.70 μM 3.33mg / L ZnSO ₄ · 7H ₂ O 5.22 μM 1.50mg / L Furtherance density

Vitamins
Glycine 26.64 μM 2.00mg / L Thiamine-HCl 2.96 μM 1.00mg / L Pyridoxine-HCl 2.43μM 0.50mg / L Nicotinic acid 4.06μM 0.50mg / L

Thereafter, root tissues containing the division delay center were collected from the plants germinated for about 5 to 6 days. Root canals were removed from the roots and 1 mm sections from the cuts were obtained as a section.

1-2: Fission Delay Center  Induction and Isolation of Derived Cell Lines

(1) The fragments obtained in Example 1-1 were separated from each N6 medium containing 2 mg / L, 3 mg / L, and 4 mg / L of 2,4-D, which was a different type of auxin hormone, IAA. (Indole-3-acetic acid), IBA (Indole-3-butyric acid), NAA (1-naphthaleneacetic acid), CPA (p-chlorophenoxyacetic acid), picloram (Picloram, 4-amino-3,5,6 -trichloropicolinic acid) was inoculated in N6 medium containing the same concentration.

As a result, induction of cell line was observed at 30 days after inoculation in the medium containing 2,4-D. The medium containing 2,4-D did not increase the induction rate of the cells derived from the delayed delay center in proportion to the concentration, and the induction rate was similar at the concentration of 2 mg / L and higher. Therefore, it was found to be preferable to culture at a concentration of 2mg / L or more. However, as a result of further experiments for 1mg / L, 4mg / L, 5mg / L, 6mg / L and 7mg / L it was found that it is more preferable to incubate at a concentration of 2 ~ 7mg / L.

On the other hand, as shown in Figure 2, except for 2,4-D auxin, cells were not induced after inoculation, but the roots were generated in the explants. In FIG. 2, A was incubated with 2,4-D, B was CPA, C was IAA, D was IBA, E was NAA, and F was incubated with picloram.

Therefore, it could be seen that the cell line derived from the division delay center specifically induced when 2,4-D was used.

(2) As shown in FIG. 1A, morphological differences were observed between root tissues other than the division delay center and cell lines derived from the division delay center when cultured in a medium containing 2,4-D. In other words, heterogeneous and local differentiation was observed in root tissue-derived cell lines, but division-centered cell lines were homogenous and no local differentiation was observed. When visually observed, the cell lines derived from root tissues other than the cleavage delay center were yellowish, whereas the cell lines derived from the cleavage delay center were white and surrounded by mucous substances. On the basis of these morphological differences, the tissue isolated with white coloration, that is, the cell line derived from the mitotic retardation center, was isolated after 3 to 6 weeks. In FIG. 1A, the red circle is a cell line derived from a division delay center.

(3) FIG. 3 is a photograph showing a cell line (a) derived from the root tissue other than the lysis center and a cell line (b) derived from the lysis center. . As shown in Fig. 3, heterogeneous and local differentiation was observed in the root tissue-derived cell line, but the cell line derived from the mitotic retardation center was homogeneous and no localized differentiation was observed.

(4) On the other hand, when the cell line derived from the dissociation delay center was mixed with the cell line derived from the root tissue other than the dissociation delay center, the mucous component was denatured without being separated for about 3 to 6 weeks, It is believed that this is due to the phenolic compounds in the cell line. This is because the synthesis of phenol components is actively performed in differentiated tissues.

Example  2. of corn Fission Delay Center  Isolation of Derived Cell Lines

Corn seeds were germinated in the same manner as in Example 1-1, and fragments containing the division delay center were obtained from the roots of the germinated plants. Then, in the same manner as in Example 1-2, by incubating the fragments in a medium containing 2,4-D, CPA, IAA, IBA, NAA, and picloram, respectively, whether a cell line derived from a division delay center is induced. Observed.

As a result, as shown in Figure 4, in the case of culturing the fragment containing the delayed core portion of the corn, the cell line in the case of the medium containing 2,4-D as in the case of inducing the root fragment containing the split delay center of the rice Induction was observed, but in the case of other auxins except 2,4-D, the cells were not induced after inoculation, but the root muscle was generated in the explants. In FIG. 4, A is cultured with 2,4-D, B is CPA, C is IAA, D is IBA, E is NAA, and F is a picloram.

Therefore, it was confirmed that 2,4-D-specific cell lines derived from the mitotic retardation center can be induced in plants containing the cleavage delay center other than rice.

Example  3. of rice Fission Delay Center  Observation of Derived Cell Lines

3-1: Observation of morphological changes in long term culture

The cleavage delay center-derived cell line isolated in Example 1 was grown by inoculating into a culture medium having the same composition as the cell line induction step of Example 1-2, that is, N6 medium containing 2 mg / L of 2,4-D. . After 4 and 16 weeks of proliferation, the proliferating divisions of central division cells were observed. In addition, as a control group, cell lines derived from root tissues other than the division delay center were also grown in the same manner, and then morphological changes of the cells were observed.

As a result, as shown in Figure 5, the morphological variation was observed after 4 weeks of culture (A) and 16 weeks of culture (except photographs A) in the root tissue-derived cell line. After 16 weeks of culture, several local differentiation was observed in the cell mass.

On the other hand, as shown in Figure 6, when cultured cell lines derived from division delay centers did not occur morphological variation even after 4 weeks (A) and 16 weeks (B) culture.

Therefore, it was confirmed that the cell line derived from root tissue other than the center of cleavage delay showed a morphological variation with time, but the cell line derived from the cleavage delay center showed morphological stability. As described above, the cell line derived from the mitotic retardation center according to the present invention is stably maintained in a long-term culture with a cell line of unicellular origin, so that it is highly desirable to select a cell line having a high yield and stable substance production ability.

On the other hand, the cell line derived from the isolated cleavage delay center had a large nucleus morphologically. As shown in FIG. 7A, the size of the cell line itself was about 10 to 20 μm, and the nucleus size was about 2 to 4 μm. On the contrary, as shown in Fig. 7B, the root-tissue-derived cell line showed a very small nucleus size as compared with the cell line derived from the mitotic retardation center.

3-2: Suspension Culture  Observe cell aggregation

Unlike microbial cells, plant cells are cultured in the form of cell masses rather than single cells, and are cultured in as few as several hundreds. This cell aggregation causes environmental differences between the inside and outside of the cell mass, which leads to mutations in cell proliferation and useful material production. Therefore, the suspension of the cell lines derived from the division division center and cell lines derived from root tissues other than the division division center were observed for cell aggregation.

As a result, as shown in FIG. 8 and FIG. 9, it was confirmed that the cell line derived from the root tissue other than the cleavage delay center was cultured in the form of aggregates of several hundred cells, but the cell of the delayed cell division was cultured as a single cell. Fig. 8 is a photograph showing the cell levels of the root tissue-derived cell line (a) and the lysis center-derived cell line (b) other than the divisional delay center. Fig. 9 is a photograph showing the aggregation of the root- Is a graph showing the rate.

3-3: Survival rate test during cryopreservation

Cryopreservation technology in cell lines is an essential way to ensure a stable supply of raw materials and to establish a practical master cell bank. Cryopreservation techniques are widely used in animal cells, but plant cells are known to have limited application range due to low survival rate after cryopreservation. Thus, the cryopreservation survival of the cells obtained from the division delay center of the present invention was tested as follows.

The suspension culture of Example 3-2 was inoculated and incubated for 6 to 7 days. The suspension culture was preliminarily cultured for 3 days in a medium containing 0.16 M of mannitol at room temperature. Lt; / RTI > The cold-treated cells were harvested and transferred to a cryobial (Duran, USA) medium containing 40% ethylene glycol (Sigma, USA) and 30% sorbitol (DUCHEFA, The Netherlands) And cultured for 3 minutes. Thereafter, the cultured cells treated with the cryopreservative were immersed in liquid nitrogen and frozen. After that, cultured cells kept in liquid nitrogen for 10 minutes or longer were taken out for thawing, and placed in a water bath at 40 ° C for 1 to 2 minutes. For cell regeneration, the filtrate containing the cells was applied onto a 0.5 M sorbitol supplemented and stabilized at room temperature for 30 minutes. Then, the cells were incubated in medium containing 0.1 M sorbitol for 24 hours, in sorbitol-containing medium for 24 hours, and in sorbitol-containing medium for 24 hours, and cell viability was observed.

As a result, as shown in Figure 10 and 11, the root tissue-derived cell line showed a survival rate of less than 10%, the division of the division delay center-derived cell line of the present invention showed a survival rate of about 85% or more. FIG. 10 is a photograph illustrating survival rates of cell lines derived from root tissues (a) and cell lines derived from division delay centers (b) other than the division division center through Evan's Blue staining after cryopreservation, and FIG. 11 is a graph showing this.

Therefore, it was confirmed that the cell line obtained from the division-retarding center part of the present invention can be frozen and stored, while the normal plant cell has a low survival rate upon freezing and thus the cell line can not be stored by the method of cryopreservation. Thus, it was confirmed that the cell line derived from the mitotic retardation center is a preferable form for long term preservation.

The specific parts of the present invention have been described in detail above, and for those skilled in the art, these specific descriptions are merely preferred embodiments, and the scope of the present invention is not limited thereto. Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (2)

Cell lines derived from the divisional delay center of the plant, derived from the divisional delay center of the plant and having the following characteristics:
(a) present as single cell in suspension culture;
(b) exhibits morphological features with larger nuclei compared to cell lines derived from tissues other than the division delay center;
(c) surrounded by mucous material;
(d) remain stable without morphological variation in long term culture; And
(e) High survival rate in cryopreservation.
The cell line of claim 1, wherein the plant is selected from the group consisting of rice, corn, peas, oats, onions, and Arabidopsis.

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