KR101963644B1 - Method of Cultivating Youngia denticulate using Plant Tissue Culture Techniques and Cosmetic Composition containing an Extract from Callus or Suspension Culture - Google Patents

Abstract

(A) disinfecting the seeded and depleted seeds; (b) seeding and sterilizing the disinfected seeds, and (c) seeding the cotyledons of the germinated seeds with naphthaleneacetic acid (NAA), benzyladenine (BA) or To a medium supplemented with the above-mentioned mixture, followed by tissue culture of the squeezing.
According to the present invention, it is possible to increase the efficiency of the propagation of the high quality seedlings required in the mass production process of the seeds and to improve the quality of the plant tissues Establishing seed sterilization conditions for in-vitro cultivation of seedlings so as to establish a mass propagation system using the cultivation technique and to develop a large-scale propagation system. It is possible to establish an induction condition for undifferentiated cell tissue having a re-differentiation ability, and to establish an optimum culture medium for inducing a regenerated plant from undifferentiated plant cells.
In addition, an extract having excellent antioxidative effects can be obtained from the callus and the culture medium which are tissue-cultured by the minus tissue culture method of the present invention and can be used for the production of a cosmetic composition.

Description

TECHNICAL FIELD [0001] The present invention relates to a cosmetic composition containing as an active ingredient an extract derived from callus or suspension culture, Suspension Culture}

The present invention relates to a cosmetic composition containing as an active ingredient an extract from a callus or a suspension culture and a method for mass production of the extract through an in-vitro culture, and more particularly to a cosmetic composition comprising a genetically modified crop, Development of growth-promoting varieties and re-breeding ability for the development of large breeding system through in-vitro cultivation. Extraction of large quantities of production methods and extracts from the callus and in-vitro culture media. ≪ / RTI >

Callus is an undifferentiated irregular cell mass that is typical of tumor tissues formed by cleavage tissue around the wound when the plant is injured. Plants are composed of dividing tissues that are largely dividing cells and permanent tissues. When cells of the first dividing line are put in nutrient medium, callus is formed. Thereafter, males are formed and differentiated into plants. This callus is also often called the stem cell of a plant.

Various tissue culture techniques have been applied and developed in some specialized medicinal plants such as ginseng. However, there have been no studies on in vitro culture technology for mass propagation of the extracts. The development of tissue culture technology in previous horticultural crops was carried out with the aim of cultivating root tissue (for example, ginseng adventitious roots) for industrial use or inducing regeneration for research use.

For the industrialization of high value-added functional natural material, it is important to establish an economical production system, high quality of natural material, and high added value. And deprivation was reported as a bioactive activity related to liver function recovery through previous studies and it is a promising functional natural material that goes to the stage of industrialization through technology transfer to pharmaceutical companies.

Stable securing of natural seedling plants is the first condition to go to the mass production system. In the case of micropropagation using plant tissue culture technology, uniform quality seeds are grown under aseptic conditions in large quantities, It is a plant propagation technology that can produce stable without. Establishment of a micro breeding system has the difficulty of individually establishing optimal culture conditions suitable for different plant species. However, once the condition is established, it can be advantageously used for various subsequent studies using the plant material. The large-scale breeding techniques of crops can effectively replace seed-seeding and seeding, germination and seedling management systems, which require a large amount of labor and agricultural cultivation techniques, and are effectively linked to plant plant growing systems using nutrients or artificial soil It is a technology that can be used to establish a functional natural product production system. In addition, tissue culture technology can be used to develop good varieties using various biotechnology techniques. For the flow rate through the chromosome hippocampus and the cultivation of draining habits, establishment of tissue culture technology for induction of regeneration plant should precede. Therefore, the present invention has been carried out in order to establish the induction conditions of aseptic seed germination, in vitro culture of undigested seeds, rooting and regeneratable undifferentiated cell organisms from the subtracted cotyledons, and ultimately new regeneration complete bodies.

Korean Patent Registration No. 10-1209574 Korean Patent Registration No. 10-1236233

DISCLOSURE Technical Problem Accordingly, the present invention has been made in order to solve the above problems, and it is an object of the present invention to improve the efficiency of propagation of high quality seedlings required in the mass production process of grains, to study the gene function through genetic modification, The purpose of this research is to develop a technology for cultivating and extracting large quantities of seeds using plant tissue culture technology, which can be utilized for the development of varieties.

Another object of the present invention is to establish seed sterilization conditions for in vitro culture of seedlings, for the study of vegetative tissue culture of seedlings and seedlings, to induce undifferentiated cell tissues with roots and re- Establishing conditions, and establishing optimal media composition to induce regenerated plants from undifferentiated plant cells.

Another object of the present invention is to extract a useful substance of antioxidant effect from the callus and the culture liquid cultured through the subtractive tissue culture technique and to use the extract in the production of functional cosmetic composition.

Disclosure of Invention Technical Problem [8] The present invention provides a method for the above-mentioned purpose, comprising the steps of: (a) disinfecting seeded seeds; (b) seeding and sterilizing the disinfected seeds, and (c) seeding the cotyledons of the germinated seeds with naphthaleneacetic acid (NAA), benzyladenine (BA) or To a medium supplemented with the above-mentioned mixture, and then tissue-culturing the depilated tissue.

Further, in step (c), it is preferable to induce the root tissue to be in the form of an improved plant by culturing in a medium supplemented with 1 to 10 mg / L of naphthaleneacetic acid (NAA) .

Also, in the step (c), the culture medium containing 0 to 3 mg / L of naphthaleneacetic acid (NAA) and 4 to 20 mg / L of benzyladenine (BA) Followed by induction in the form of regeneration shoots.

In addition, in step (c), the culture medium may be prepared by adding 0.5 to 7 mg / L of naphthaleneacetic acid (NAA) and 5 to 20 mg / L of benzyladenine (BA) Followed by culturing, in the form of callus.

The callus fraction induced after the step (c) was added to a medium supplemented with 0.5 to 1.5 mg / L of naphthaleneacetic acid (NAA) and 5 to 15 mg / L of benzyladenine (BA) And culturing the callus extract.

In step (a), the seeds are dipped in 70% ethanol, suspended in a suspension, suspended in a 10% chlorox solution, washed with sterilized water, and germinated aseptically.

In order to achieve the above object, the present invention also provides a cosmetic composition comprising the presently delivered extract as an active ingredient, which is extracted from callus or suspended culture medium.

According to the present invention, it is possible to increase the efficiency of the propagation of the high quality seedlings required in the mass production process of the seeds and to improve the quality of the plant tissues Establishing seed sterilization conditions for in-vitro cultivation of seedlings so as to establish a mass propagation system using the cultivation technique and to develop a large-scale propagation system. It is possible to establish an induction condition for undifferentiated cell tissue having a re-differentiation ability, and to establish an optimal culture medium for inducing regenerated plant from undifferentiated plant cells.

In addition, an extract having excellent antioxidative effects can be obtained from the callus and the culture medium which are tissue-cultured by the minus tissue culture method of the present invention and can be used for the production of a cosmetic composition.

FIG. 1 is an image showing representative tissue culturing entities derived on NAA and BA mixed treatment media at different concentrations for establishing the subtracted roots, regenerating undifferentiated tissue and regeneration shoot initiation conditions.
FIG. 2 is a photograph showing roots induction and cultivation images of regenerated plants according to an embodiment of the present invention. FIG.
FIG. 3 shows the result of measuring the DPPH radical scavenging ability of the culture solution according to the example of the present invention every two weeks.
FIG. 4 shows the result of measuring the ABTS radical scavenging ability of the culture solution according to the embodiment of the present invention every two weeks.
FIG. 5 is a graph showing the DPPH radical scavenging activity of callus of the culture solution according to the example of the present invention every 2 weeks,
FIG. 6 shows the result of measuring the ABTS radical scavenging ability of the culture solution every 2 weeks according to the embodiment of the present invention.
FIG. 7 is a graph showing a change in weight of a callus of a culture solution according to an embodiment of the present invention. FIG.

(A) disinfecting the seeded and depleted seeds; (b) seeding and sterilizing the disinfected seeds, and (c) seeding the cotyledons of the germinated seeds with naphthaleneacetic acid (NAA), benzyladenine (BA) or To a medium supplemented with the above-mentioned mixture, followed by tissue culture of the squeezing.

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

Regarding the method of producing the seeds by the above-mentioned tissue culture, the regeneration type of the regenerated plant can be controlled by regulating the content of the plant hormone in the medium composition for suspension culture of the callus in the regeneration plant cultivation.

Namely, in the step of damaging the cotyledons of the nipple of the above-mentioned subtraction, it can be induced in the form of a plant having developed root tissue upon culturing in a medium supplemented with 1 to 10 mg / L of naphthaleneacetic acid (NAA) And can be induced in the form of regenerated shoots when cultured in a medium supplemented with 0 to 3 mg / L of naphthaleneacetic acid (NAA) and 4 to 20 mg / L of benzyladenine (BA) Can be induced in the form of callus when cultured in a medium supplemented with 0.5 to 7 mg / L of naphthaleneacetic acid (NAA) and 5 to 20 mg / L of benzyladenine (BA).

In addition, the inducible callus can be mass-produced by mass-producing a useful substance by culturing it without directly differentiating it into an individual. In other words. The callus fraction induced after the step (c) was suspended in a medium supplemented with 0.5 to 1.5 mg / L of naphthaleneacetic acid (NAA) and 5 to 15 mg / L of benzyladenine (BA) It is possible to cultivate and mass-extract callus.

Accordingly, another embodiment of the present invention relates to a cosmetic composition comprising an extract obtained from the extract and a cosmetic composition containing the extract as an active ingredient.

In addition, in the case of (a), the seeds were immersed in 70% ethanol, suspended in water, and sterilized for 10 days. Suspension in% chlorox solution and sterile germination after sterilization were the most effective to minimize contamination.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

[Example]

1. Establishing the conditions for aseptic germination

In order to establish germination condition of sterilized cabbage seeds, 5, 10, 15 and 20 minutes of washing in 70% ethanol and 10% chloroxic solution showed seed fungal contamination when washed for 5 ~ 15 minutes. The germination of normal plants could be confirmed while the seed contamination was minimized by washing for 20 minutes.

2. Establish conditions for regrowth and regrowth.

FIG. 1 is a graph showing the effect of different concentrations of naphthaleneacetic acid (NAA) and benzyladenine (Benzyl adenine, BA) on induced growth of undigested roots, regenerating undifferentiated tissues and regeneration shoot initiation conditions This is an image showing the culture representative picture.

The seedlings germinated in aseptic conditions and seeded in unfiltered seedlings were seeded on MS medium containing different concentrations of NAA and BA to establish rooting induction, greenish callus type induction, and regeneration. And cultured for 16 hours / day for 5 weeks. The experiment was carried out with 3 replicates per treatment medium, 9 sections per each culture condition, and the slices were subcultured into fresh regeneration medium at the same temperature condition at 24 ± 2 ℃ for 2 weeks. The growth of roots was most effectively induced in media containing 1 to 5 mg / L NAA, and the induction of redish-capable greenish callus was induced by high-concentration BA (10-20 mg / L) , And induction of regrowth shoots was effectively induced in medium containing low concentration NAA and high concentration BA (Fig. 1).

As a result of measuring the number of roots derived from subtracted cotyledons at different treatments, the highest number of roots per section was found in the medium containing 1 mg / L of NAA and 0 mg / L of BA, (20 mg / L) treatment (Table 1). These results indicate that the medium conditions containing 1 mg / L of NAA are most effective for roots cultivation and subtraction, and the morphological aspect of the derived roots is the result of the treatment of high-concentration NAA (10 mg / L) As shown in the induction results, low concentration of NAA alone was effective for root cultivation.

Table 1 shows the results of measuring the number of roots induced on NAA and BA mixed treatment media at different concentrations.

NAA (mg / L) 0 One 5 10 20

BA (mg / L)
0 0.2 ± 0.1 2.3 ± 0.1 1.7 ± 1.2 2.0 ± 0.3 0.5 ± 0.3 One 0.0 ± 0.0 0.4 ± 0.2 0.4 0.4 0.3 ± 0.2 0.0 ± 0.0 5 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.1 ± 0.2 0.0 ± 0.0 10 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 20 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0

The massive organism of the greenish callus type with re-differentiation potential is an essential condition for large-scale plant propagation through the application of subtractive tissue culture technology. In this experiment, the presence of greenish callus was confirmed in media containing high concentration BA (10-20 mg / L) (FIG. 1), and the number of greenish callus was measured. As a result, no induction of greenish callus was observed in BA- , Higher NAA concentration, and lower NAA (1-5 mg / L) than NAA treatment (Table 2). The highest number of greenish callus induction conditions were found to be effective for greenish callus mass organisms in medium containing 1 mg / L NAA and 20 mg / L BA, including low NAA and high concentration of BA.

Table 2 shows the results of measuring the number of re-differentiating greenish callus derived from different concentrations of NAA and BA mixed treatment media.

NAA (mg / L) 0 One 5 10 20

BA (mg / L)
0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 One 0.4 ± 0.1 1.3 ± 0.2 1.1 ± 0.8 0.5 ± 0.1 0.0 ± 0.0 5 0.2 ± 0.0 4.0 ± 2.6 1.3 ± 0.7 2.4 ± 1.8 0.0 ± 0.0 10 0.5 ± 0.6 3.9 ± 3.2 4.7 ± 0.8 0.0 ± 0.0 0.0 ± 0.0 20 0.3 ± 0.3 8.3 ± 4.8 3.3 ± 2.9 1.7 ± 0.2 0.8 0.8

The differentiation of NAA and BA by mixing different concentrations of NAA and BA, and the subtraction regeneration system induction, were carried out with medium composition containing 0 mg / L NAA and 20 mg / L BA, and 1 mg / L NAA and 5-20 mg / L BA (Fig. 1). The number of regenerated shoots was measured and it was confirmed that 2.3 regenerants per section were derived from medium containing 0 mg / L NAA and 20 mg / L BA (Table 3). Although the highest efficiency regeneration body induction was found in medium containing 0 mg / L NAA and 20 mg / L BA, it was observed that the induction of a large number of small shoot bundles proceeded under the above conditions, In the medium containing 1 mg / L NAA and 5 mg / L BA, the number of derived regeneration bodies was 1.2, not the highest efficiency. However, the induced regeneration shoot shape was close to the complete plant, , The above conditions were considered to be the most ideal. The regeneration shoots obtained from this experiment were rooted in 1/2 MS medium for root induction and complete plant cultivation. Normal roots development and leaf growth are now observed, and the in-vitro culture conditions established in this experiment are It was confirmed to be effective for regeneration (Fig. 1).

Table 3 shows the results of measurement of the number of regeneration shoots induced on NAA and BA mixed treatment media at different concentrations.

NAA (mg / L) 0 One 5 10 20

BA (mg / L)
0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 One 0.3 ± 0.3 0.1 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 5 0.0 ± 0.0 1.2 + 1.7 0.0 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 10 0.9 ± 1.4 1.0 ± 0.9 0.2 ± 0.3 0.0 ± 0.0 0.0 ± 0.0 20 2.3 ± 4.0 1.7 ± 2.2 0.03 0.1 0.0 ± 0.0 0.0 ± 0.0

Fig. 2 is a photograph showing roots induction and cultivation images of regenerated plants.

3. Establishing suspension conditions for callus suspension

(1) Put 70 ml of MS medium into a 250 ml Erlenmeyer flask and sterilize at 121 ℃ for 15 min.

(2) Callus is cut into 1 mm in diameter, and 20 callus pieces are collected and placed in a sterilized medium. At this time, the established callus induction hormone conditions, NAA 1 mg / L and BA 10 mg / L, are added.

(3) Culture at 26 DEG C, 200 rpm, 16 hr light shaker / 8 hr dark state. Change medium every two weeks - Measure the antioxidant capacity of the culture medium and callus in the medium change every 2 weeks.

FIG. 3 shows the result of measuring the DPPH radical scavenging activity of the culture solution every two weeks, and FIG. 4 shows the result of measuring ABTS radical scavenging activity every two weeks.

FIG. 5 shows the result of measuring the DPPH radical scavenging activity of the callus every 2 weeks, and FIG. 6 shows the result of measuring ABTS radical scavenging activity every 2 weeks.

As can be seen from FIGS. 3 to 6, the antioxidative activity was high in the culture medium and the culture medium (callus) of the samples 12, 13 and 14 of the examples, and the antioxidative activity of the culture medium was higher than that of the culture medium. In addition, antioxidant activity was maintained in both cultures and cultures during 2, 4, and 6 weeks of suspension culture, indicating that the antioxidant activity was maintained along with the quantitative increase of cultures during the 6 - week suspension culture period.

In general, in the case of suspension culture, since the culture medium is exchanged by exchanging the liquid medium at regular intervals, the activity or quality of the culture medium is often deteriorated when the medium is exchanged. According to the method according to the embodiment of the present invention, And the activity of the cultured organism is maintained for at least 6 weeks. Thus, it can be proved that the activity is maintained even in the long-term culture.

Table 4 shows the results of subtracting callus weight and state change.

term
Initial Callus Weight (mg) Weight of callus in mg (mg) Primary Secondary Third Primary Secondary Third 0 days 145.6 156.6 174.6 6 days 151.6 140.2 123.6 263.5 409.7 308.4 12th 127.6 100.3 99.9 1096.6 850.4 757.9 18th 95.2 79.9 95.2 1780 1740.9 2174.3 24th 104.3 94.6 105.2 4600 2807.8 4168.8 30 days 106.7 131.1 113.3 5146 4976.6 5757.4

FIG. 7 is a graph showing weight changes of the above and subtracted calluses.

As can be seen in Table 4 and FIG. 7 above, the weight of callus is steadily increased in proportion to the period by the current distribution of callus.

Claims (7)

(a) disinfecting seeds that have been seeded and removed;
(b) seeding and germinating the disinfected and depleted seeds; and
(c) cotyledon of nursery seeds of the germinated seeds onto a medium to which naphthaleneacetic acid (NAA), benzyladenine (BA), or a mixture thereof is added, and subtracting tissue culture
Lt; RTI ID = 0.0 > a < / RTI > mass production method.
The method according to claim 1,
In the step (c), the plant is cultured in a medium supplemented with 1 to 10 mg / L of naphthaleneacetic acid (NAA) during the seedling culture, thereby inducing the root tissue to be in the form of an improved plant Through in-flight culture and mass production methods.
The method according to claim 1,
In step (c), the cells are cultured in a medium supplemented with 0 to 3 mg / L of naphthaleneacetic acid (NAA) and 4 to 20 mg / L of benzyladenine (BA) , And regeneration shoots in the form of shoots.
The method according to claim 1,
In step (c), culturing is carried out in a medium supplemented with 0.5 to 7 mg / L of naphthaleneacetic acid (NAA) and 5 to 20 mg / L of benzyladenine (BA) , And callus. ≪ RTI ID = 0.0 > 8. < / RTI >
5. The method of claim 4,
The callus fraction induced after the step (c) was suspended in a medium supplemented with 0.5 to 1.5 mg / L of naphthaleneacetic acid (NAA) and 5 to 15 mg / L of benzyladenine (BA) Cultivating and culturing the callus. The method of mass-production by in-vitro culture.
The method according to claim 1,
In step (a), the seeds are dipped in 70% ethanol, suspended in a suspension, suspended in 10% chlorox solution, and washed with sterilized water for aseptic germination. Production method.
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