KR101734992B1 - A media composition for maximized anti-oxidant capacity and growth in perilla leaf induced callus, and cultivation methods thereof - Google Patents

Abstract

The present invention relates to a culture medium for increasing growth induction and anti-oxidative activity of Perilla frutescens var. japonica Hara. callus, and a culture method of Perilla frutescens var. japonica Hara. using the same and, more specifically, to a culture medium which improves callus induction and growth of Perilla frutescens var. japonica Hara. callus and can induce Perilla frutescens var. japonica Hara. callus having excellent anti-oxidative ability, and to a method for cultivating Perilla frutescens var. japonica Hara. callus using the same.

Description

Technical Field [0001] The present invention relates to a culture medium for growth induction and antioxidative ability of callus leaf callus, and to a culture method of perilla leaf induced callus and cultivation methods using the same,

The present invention relates to a culture medium for growth induction and antioxidative activity of callus leaf callus and a culture method using the same. More particularly, the present invention relates to a culture medium capable of inducing callus induction and growth of sesame leaf, And a method for culturing callus leaf using the composition.

( Perilla frutescens var. Japonica Hara.) Is a perennial herb that belongs to the family Lamiaceae (Labiatae) and has been cultivated for long time in Korea, China, and Japan for oil production (Chae et al., 1991). In Korea, it began to be cultivated from the Unified Silla period, and recently it is mainly grown in the greenhouse and greenhouse. The leaves of perilla seeds are commonly called sesame leaves, seeds of sesame seeds are used as oil for cooking oil, and also used directly as dried powders for seasoning dishes. In addition, sesame leaf is mainly grown for use as a vegetable and for breeding to eat meat cooked with seasoning and spice ingredients. The seed production area of sesame leaf in Korea is about 24,000 ha ( Agricultural & Forestry Statistical Yearbook 2006, p. 319), while the area for vegetable cultivation is estimated at about 850 ha.

Perilla is rich in essential amino acids, vitamins, and minerals as well as fat, and consumption is increasing as perilla tea and health food. In recent years, with the increase of meat consumption, sesame leaves are increasingly consumed as fresh leaves, increasing the cultivation area and contributing to the increase of farm income. The sesame leaf contains more than twice as much iron as spinach and contains nutrients such as minerals such as calcium and vitamins A and C in abundance. It inhibits the growth of M. tuberculosis and contains a large amount of SOD (Superoxide Dismutase). It is highly valued as a functional vegetable.

Recently, a plant callus material called a stem cell of a plant has been attracting attention as a raw material for food materials and cosmetics having an excellent physiologically active effect. It is clear that such a plant callus is an environmentally friendly high-tech material capable of cultivating and mass-producing by applying the cutting-edge plant tissue culture technology, but it is difficult to develop various plant callus materials due to limitations in technology development.

Currently developed callus materials include tulip bulb tissues (Korean Patent Laid-open Publication No. 10-2016-0010064), peanuts (Korean Patent Laid-Open No. 10-2016-0001961), sea urchins (Korean populations) -2016-0023034). However, as described in the present invention, there is no known a culture composition which can induce callus induction and growth of a sesame leaf and induce callus leaf having excellent antioxidant ability.

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above-mentioned needs. It is an object of the present invention to provide a 2,4-D (2,4-dichlorophenoxyacetic) plant which regulates the growth of plant cells to establish optimal conditions for improving the induction and growth of callus leaf callus. The present invention is intended to provide a medium composition for inducing growth of callus leaf, which contains as an active ingredient, medium C supplemented with an acid, potassium nitrate, and myo-inositol.

In addition, the present invention relates to a culture composition capable of inducing callus in order to develop safflower callus material having excellent antioxidant ability, and a method of culturing safflower callus having excellent antioxidative ability through the extraction process and antioxidant ability investigation of the callus leaf derived from the callus leaf .

In order to solve such a technical problem, the culture composition for inducing growth and antioxidant ability of callus leaf callus according to the present invention comprises

, And medium C to which 2,4-D (2,4-dichlorophenoxyacetic acid), potassium nitrate, and myo-inositol are added as an active ingredient.

Preferably, the medium C may include 0.02 to 0.04% by weight of 2,4-D (2,4-dichlorophenoxyacetic acid), potassium nitrate may be included in an amount of 70 to 85% by weight, Myo-inositol may be included in an amount of 2.5 to 3.5% by weight.

Preferably, the culture medium has a pH of 5.0 to 7.0, and the culture medium according to the present invention further comprises agar, sucrose, MOPS, and the like.

In addition, the method for cultivating callus leaf callus according to the present invention is characterized by including a step of inducing callus by chewing and sterilizing a sesame leaf sterilized in the medium composition.

According to the culture composition for inducing growth and antioxidative capacity of callus leaf callus according to the present invention having the above-described constitution, it is possible to provide a culture composition having optimal conditions for improving induction and growth of callus leaf callus, It is possible to provide a callus leaf having not only growth of callus but also excellent antioxidant ability.

In addition, the present invention has the effect of enabling the development of cutting-edge new materials that can be used in the food and cosmetic fields owing to the development of callus leaf material which is capable of culturing and mass-producing safflower callus having excellent antioxidant ability and having excellent growth and antioxidant ability.

1 is a graph showing the growth curves of callus leaf according to the composition of the medium.
FIG. 2 is a microscopic photograph of a perilla leaf and a callus leaf; FIG. 2 (A) is a sepal leaf; FIG. 2 (B) is a microscopic photograph of a perilla leaf callus cultured in MS modified medium; to be.
FIG. 3 is a graph comparing antioxidant activities through Oxygen Radical Absorbance Capacity (ORAC) of perilla leaf callus derived from perilla leaves and media.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which exemplary embodiments of the invention are shown. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. And this does not mean that the technical idea and scope of the present invention are limited.

According to one embodiment of the present invention, in order to induce callus leaf callus, in the present invention, the perilla leaf of the perilla ( Perilla frutescens var. Japonica Hara.) Variety is prepared by disinfection.

First, the sesame leaves are sterilized in salicylic acid solution for 1 to 3 minutes, then immersed in 70% ethanol for 5 to 20 seconds, then taken out and immersed in 1% lactic acid solution for 5 to 20 seconds to sterilize the surface and sterilized for 10 to 20 minutes Sterilized and sterilized sesame leaves are prepared, but the present invention is not limited thereto. If necessary, the sterilization process may be repeated several times.

Disinfection of sesame leaves is disadvantageous in that if the time is long, the tissue is destroyed. If the time is short, the various fungi and bacteria of the surface tissue may not be completely disinfected and contamination may occur. Do. According to one embodiment of the present invention, sterilization of sesame leaf is performed by disinfecting the surface with 5 mM salicylic acid solution for 2 minutes, then immersing in 70% ethanol for 10 seconds, taking out the solution, immersing in 1% It is preferable to perform the sterilization disinfection process twice, that is, the sterilization is performed three times for 15 minutes and then for 2 hours.

The term "callus" as defined in the present invention means an amorphous undifferentiated cell mass formed by tissue culture of organs, tissues and the like, and the callus has totipotency to regenerate into a complete adult.

The callus inducing medium used in the present invention may be any conventional one. For example, medium such as MS (Murashige and Skoog) medium, B5 (Gamborg et al.) Medium, LS (Linsmaier and McCown) medium, White medium, SH (Schenk and Hildebrandt) medium, WPM C (Carrot) medium. Of these, Medium C is preferably used.

In addition, the medium composition according to an embodiment of the present invention may further include other composition capable of inducing callus, for example, at least one growth regulator selected from the group consisting of auxin and cytokinin, May be added and may include agar or gellan gum or carbon sources such as sorbitol, mannitol, fructose, glucose or sucrose, and MOPS.

Examples of the growth regulator include oxine-based naphthalene acetic acid (NAA), 2,4-D (2,4-dichlorophenoxyacetic acid), indole-3-acetic acid (IAA) Indolebutric acid (IBA), and the cytokinin system may use one or more of benzyl adenine (BA), kinetin, and zeatin, According to one embodiment of the present invention, 2,4-D (2,4-dichlorophenoxyacetic acid) is preferably used as the growth regulator, and 2,4-dichlorophenoxyacetic acid (2,4-D) Is preferably contained in a concentration of 0.02 to 0.04% by weight based on the medium C. [

 In addition, the medium composition according to one embodiment of the present invention is characterized by containing medium C, in which potassium nitrate or myo-inositol is added, as an active ingredient, wherein the potassium nitrate ) Acts as a nutrient of cell growth. It usually serves as a nitrogen source of plant cells. Myo-inositol is a substance that helps cell wall growth and induces the growth and induction of callus leaf callus. It is one of the main composition to improve.

Preferably, the medium C may include 0.02 to 0.04% by weight of 2,4-D (2,4-dichlorophenoxyacetic acid), potassium nitrate may be included in an amount of 70 to 85% by weight, Myo-inositol may be included in an amount of 2.5 to 3.5% by weight. More preferably, potassium nitrate may be contained in an amount of 75 to 80% by weight, and myo-inositol may be included in an amount of 3.0 to 3.3% by weight.

Here, the pH of the culture medium composition can be adjusted to pH 5.0 to 7.0. Under these conditions, tissue culture efficiency and callus inducing effect can be excellent. At this time, it is preferable to adjust the pH to 5.5 to 6.0, more preferably to adjust the pH to 5.6 to 5.8.

The method for cultivating callus leaf callus according to an embodiment of the present invention includes the step of inducing callus by dipping a sterilized sesame leaf into the medium composition and culturing at room temperature for 4 to 6 weeks.

At this time, if the cell of the tissue divides into cells and a callus, which is a mass of cells, is formed, the callus leaf alone may be separated and cultured on a new medium of the same condition as the medium composition.

The callus of callus leaf thus obtained can be provided not only as an excellent growth ability but also as an antioxidant, and as an improved callus. Further, since the development of callus leaf material having excellent growth ability and antioxidation ability, Can be developed.

In another embodiment of the present invention, the callus leaf culture may be contained in the cosmetic composition in the form of an extract after being decompressed and concentrated, or in a powder form after lyophilization. Preferably, the culture of callus leaf may contain 1 to 50% by weight based on the total weight of the cosmetic composition. In this range, the cosmetic composition is harmless to the human body and has excellent formulation stability, and is excellent in skin moisturizing, antioxidant, It is excellent in aging effect.

The cosmetic composition containing the callus leaf culture of the present invention may be formulated into cosmetic formulations such as solutions, gels, creams, lotions, powders, lotions, foundations or solidified soaps, but is not limited thereto. In general, the general components used as a raw material of a cosmetic composition can be used for the production of a specific formulation. Specific examples thereof include solvents, solubilizers, moisturizers, gelling agents, thickeners, softeners, stabilizers, emulsifiers, surfactants, antioxidants, , Bactericides, vitamins, fragrances, oils, dyes and the like can be added and can be appropriately selected by those skilled in the art.

In addition to the components generally contained in the cosmetic composition containing the callus leaf culture according to one embodiment of the present invention, other components which can be used singly or in combination with other components may be included within the scope of not affecting the effect of the present invention Of course.

[Example]

Hereinafter, the present invention will be described in more detail with reference to specific examples.

Example 1 Preparation of callus induction

1. Sesame leaf sterilization

According to the present invention for the induction of callus sesame perilla (Perilla I have frutescens . japonica Hara.) Prepare the sesame leaf of the breed by disinfection.

First, the sesame leaves were sterilized on a 5 mM salicylic acid solution for 2 minutes, then immersed in 70% ethanol for 10 seconds, then taken out and immersed again in a 1% lactic solution for 10 seconds, the surface was sterilized and washed three times with sterilized water for 15 minutes. Dry for a period of time. Dried sesame leaves repeat this sterilization process once again.

2. Prepare callus induction medium

MS (Murashige and Skoog) meiam (w / Vitamins), MS (Murashige and Skoog) modified medium and Medium C are prepared as shown in Table 1 below.

At this time, Medium C contains 0.0313 wt% of 2,4-D (2,4-dichlorophenoxyacetic acid), 78.3579 wt% of potassium nitrate, 3.1343 wt% of myo-inositol, .

 Primary medium ingredient table Name MS Medium
(w / Vitamins)  MS Modified Medium  Medium C Content Content Content Ammonium nitrate 37.407% 4.7636%  4.2% Boric acid 0.141% 0.0179%  0.094% Calcium chloride, anhydrous 7.531% 0.9591%  3.5493% Cobalt chloride 6H ₂ O 0.001% 0.0001%  0.0008% Cupric sulfate 5H ₂ O 0.001% 0.0001%  0.0008% Ferric sodium EDTA 3H ₂ O 0.955% 0.1216%  1.3199% Magnesium sulfate, anhydrous 4.097% 0.5217%  3.8267% Manganese sulfate H ₂ O 0.383% 0.0488%  0.3134% _{Molybdic acid (Sodium salt) 2H 2} O 0.006% 0.0007%  0.0078% Potassium iodide 0.019% 0.0024%  0.0235% Potassium nitrate 43.074% 5.4854%  78.3579% Potassium phosphate, monobasic 3.854% 0.4908%  4.7015% Sodium phosphate, monobasic - 0.4273% - Zinc sulfate 7H ₂ O 0.195% 0.0248%  0.0627% Adenine hemisulfate - 0.231%  - Kinetin - 0.0029%  - Myo-Inositol 2.267% 0.2887%  3.1343% a-Naphthalene acetic acid (NAA) - 0.0003% - Sucrose - 86.6116% - Thiamine HCl 0.002% 0.0012% 0.3135% 2,4-Dichlorophenoxyacetic acid
(2,4-D) - - 0.0313% Nicotinic acid (Free acid) 0.011% - 0.0313% Pyridoxine HCl 0.011% -  0.0313% Glycine 0.045% - - Total 100% 100% 100%

Agar, sucrose and MOPS were added to each prepared medium to adjust the pH to 5.6 ~ 5.8, sterilized at 121 ° C for 15 minutes at high pressure, and dispensed into a petri dish to be used as callus induction medium.

The composition of agar, sucrose and MOPS added to each medium is shown in Table 2 below.

Final Culture Medium Composition Table MS Medium (w / Vitamins)  MS Modified Medium  Medium C The primary media components (Table 1) 4.41 g 34.64 g 3.19 g agar 8g 8g 8g sucrose 10.09 g 10.09 g 10.09 g MOPS 0.5 g 0.5 g 0.5 g Third distilled water The above components were dissolved in 1 L of tertiary distilled water, adjusted to pH 5.7 with 1M KOH, sterilized

3. Stimulation of callus induction

The sterilized sesame leaf tissues were cut to a size of about 0.5 ~ 1 ㎠ and placed on each callus induction medium. The callus was induced by incubation at room temperature for 4 to 6 weeks.

Example 2: Measurement of growth of callus leaf callus

Leaf callus induced in each medium was measured at 4 to 5 days intervals and the callus size was measured. The callus formed from the four corners of the leaf was measured and the mean value and standard error (SE) were obtained.

As a result, mesophyll callus was induced in all media, but induction was faster in MS modified medium and medium C than in conventional MS medium, and the induction of callus leaf and the growth rate were remarkably fast in medium C (See Fig. 1).

These results suggest that the addition of growth regulators (Kinetin, NAA, 2,4-D, etc.) contained in the culture medium, especially 2,4-D, potassium nitrate, myoinositol -inositol) added medium C showed excellent callus induction and growth, and microscopic examination showed amorphous undifferentiated cell masses not differentiated from adult leaves (see FIG. 2).

Example 3: Antioxidant activity measurement

1. Leaf callus extraction

The adult calli and calli induced from each medium were extracted with 70% ethanol in an incubator at 40 ° C for 2 hours. The extract was centrifuged (4 ° C, 8,000 rpm, 30 minutes), filtered and concentrated using a rotary evaporator at 60 ° C. The concentrated extract was lyophilized and used as a sample for antioxidant activity measurement.

2. Measurement of antioxidant capacity (ORAC (Oxygen Radical Absorbance Capacity) analysis)

ORAC analysis was carried out to investigate the antioxidant capacity of adult leaves and callus leaf. 100 μl of 80 nM fluorescein solution dissolved in 75 mM phosphate buffer and 50 μl of each diluted sample were added to each well of a 96-well black plate using phosphate buffer, and 2,2'-Azobis (2-amidino -propane) dihydrochloride (AAPH) was added to each well and fluorescence was measured for 100 cycles at 2 minutes intervals at 37 ° C using GENios fluorescence plate reader (TECAN Trading AG, Switzerland).

As a result, it was confirmed that the callus-derived extract according to the medium C composition was more than twice the antioxidant ability at all concentrations than the callus extract derived from the MS modified medium, ).

As a result, in medium C supplemented with 2,4-D, potassium nitrate and myo-inositol, the induction and growth of excellent callus leaf callus was improved and the development of callus leaf material having excellent antioxidant ability I could see that it was possible.

The callus was superior to the adult leaves in the growth factors but had low antioxidant ability. However, the callus induced by medium C induces an excellent antioxidant ability to overcome these disadvantages to some extent.

Therefore, according to the culture composition for inducing growth and antioxidative capacity of callus leaf callus according to the present invention, it is possible to provide a culture composition having optimal conditions for improving the induction and growth of callus leaf calli, And can provide a callus leaf having excellent antioxidant ability.

In addition, the present invention has the effect of enabling the development of cutting-edge new materials that can be used in food and cosmetic fields due to the development of callus leaf material which is capable of culturing and mass-producing safflower callus having excellent antioxidant ability and having excellent growth ability and antioxidant ability.

[Production Example] Preparation of a cosmetic composition containing a culture of perilla leaf callus

Creams were prepared using the raw materials described in Table 3 below to prepare cosmetic compositions containing the cultured leaves of callus cultured in the above examples.

10 parts by weight of the culture of Example 1 was added to 100 parts by weight of 70% ethanol, aged at room temperature for about one week, and then filtered to obtain a filtrate. The filtrate was concentrated under reduced pressure at 60 DEG C with a rotary evaporator to obtain a concentrated extract.

Ingredients of cream cosmetic ingredients containing callus cultures of sesame leaves Ingredients content (% by weight) Purified water 70.29 Extract of callus leaf extract of Example 1 5.0 Carboxymethylcellulose 15.0 Glyceryl stearate 2.0 Stearic acid 3.0  1,3-butylene glycol 2.0  glycerin 1.0 Cetearyl alcohol 1.0 Beads wax 0.6 Citric acid 0.1 antiseptic 0.01 Total 100

Although the present invention has been described with reference to the preferred embodiments of the present invention, it should be understood that the present invention is not limited thereto and that the present invention is not limited thereto. Various modifications may be made by those skilled in the art.

Claims (6)

In the medium composition for callus induction, Ammonium nitrate 4.2 wt%, Boric acid 0.094 wt%, Calcium chloride anhydrous 3.5493 wt%, Cobalt chloride 6H ₂ O 0.0008 _{wt%, Cupric sulfate 5H 2 O 0.0008} % by weight, Ferric sodium EDTA 3H ₂ O 1.3199 wt%, Magnesium sulfate, anhydrous 3.8267 wt%, Manganese sulfate H ₂ O 0.3134 wt%, Molybdic acid (sodium salt) 2H ₂ O 0.0078 wt%, Potassium iodide 0.0235 wt%, Potassium nitrate 78.3579 wt%, Potassium phosphate monobasic 4.7015% by weight, Zinc sulfate 7H ₂ O 0.0627% by weight, Myo-Inositol 3.1343% by weight, Thiamine HCl 0.3135% by weight, 2,4-Dichlorophenoxyacetic acid 0.0313% by weight, Nicotinic acid Free acid 0.0313% by weight and Pyridoxine HCl 0.0313% Which contains medium C as an active ingredient, for inducing growth and antioxidant activity of callus leaf callus.
delete delete The method according to claim 1,
Wherein the medium composition has a pH of 5.0 to 7.0, and wherein the medium composition is for growth induction and antioxidative activity of callus leaf callus.
The culture composition according to claim 1, wherein the culture medium composition further comprises agar, sucrose, and MOPS.
A method for cultivating callus leaf callus comprising the step of inducing callus by dipping and cultivating a sterilized sesame leaf in the culture composition of any one of claims 1, 4 and 5.

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