KR101018842B1 - Culture method of Pelargonium sp. cell - Google Patents

Abstract

The present invention relates to a method for propagating plant cells of the genus Pelargonium, and more particularly, to sterilize and inoculate Pelargonium sp. Plants to induce and proliferate cells in a specific cell growth medium; Pelargonium sp. Plant cells prepared by the above method; Inducing and propagating adventitious roots into said cells; Pelargonium sp. Plant irregular root prepared by the above method; Plant of genus Pelargonium comprising 3/4 MS medium with NH ₄ ⁺ : NO ₃ ⁻ = 15: 30, sucrose 30 g · L ⁻¹ and NAA 5.0 mg · L ⁻¹ It relates to a cell induction medium.

Pelargonium, cell, callus, proliferation

Description

Proliferation of plant cells of the genus Pelargonium {Culture method of Pelargonium sp. cell}

The present invention relates to a method for propagating plant cells of the genus Pelargonium.

Pelargonium sidoides is a perennial shrub plant of the genus Mypenaceae, with about 270 species. About 80% of them are distributed in South Africa, and have been highly regarded as traditional therapeutic drugs.

Pelargonium The mixture is extracted from the roots and tubers of sidoyi Death traditionally tuberculosis, diarrhea, dysentery, has been used for the purpose of treatment of colds and tuberculosis, Bladt, etc. (Bladt, S. (1977) Deutsche Apotheker Zeitung 177, 1655-1660) was that important plant metabolites Pella Konishi Titanium sidoyi des extract is made up of simple phenol containing coumarin (coumarin), gallic acid (gallic acid). Currently available Pelargonium sidoides root extracts ( Pelargonium , Medicherb UK; Pelargonium Syrup, Bioharmony Africa and Umckaloabo, Spitzner) have been used to treat gastrointestinal diseases of a wide range of respiratory infections, especially respiratory infections caused by infections. (AG White, MT Davies-Coleman, and BS Ripley (2008) South African journal of botany. 74: 260-267), especially in Germany, Mexico, Turkey, Brazil and South Africa.

As such, the pelargonium sidoides is popular in the European countries due to its physiological activity effect, but is limited to cultivation of open field, so a more efficient production method is required for continuous supply of stable active substances and industrial mass production. Recently, tissue culture of plants has been applied as the most effective production method for mass production of active substances of plants. Tissue culture can select genetically superior individuals to supply uniform raw materials in a short period of time without the influence of soil, climate, etc., and obtain high concentrations of pure substances. In addition, cells and organs that are cultured after induction in the cabin have the advantage of increasing the growth rate and the content of specific secondary metabolites by controlling the in-flight culture environment (Rao SR, and Ravishankar GA (2002) Biotechnol. Adv. 20 101-153). The mass production technology of ginsenosides using ginseng root muscle using this tissue culture technology has been tried by several researchers, and some are successfully progressing to industrialization (Hahn, EJ, YS Kim, KW Yu) , CS Jeong, and KY Paek (2003) J. Plant Biotechnol. 5: 1-6).

The tissues and the physicochemical properties of the medium are very important because the tissues obtain most of the nutrients necessary for growth through the medium. The requirement of nutrients necessary for cultivation varies greatly depending on the type of plant, cultured tissue and organs, so that the components of the proper medium can only be found after many experiments. Therefore, when extracting and culturing the explants from the tissue, it is important to provide a medium suitable for the characteristics of the cultured tissue. Factors affecting the growth of cells and organs cultured on board include chemical factors such as plant growth regulators, inorganic salts and carbon sources in the medium, and physical factors such as sugar, temperature, air supply, and inoculation density. (Paek, KY, EJ Hahn, and SH Son (2001) In Vitro Cell Dev. Biol. 37: 284-292). The principle that mineral salt concentration controls plant growth and secondary metabolites has been reported to influence the absorption of water and inorganic nutrients from the medium by controlling the water potential of the medium (Gueon, DY, YI Park, SS Jun, CD). Jin, and YN Hong (2001) Plant physiology.In: Hopkins WG (eds) .Introduction to plant physiology.Eulyoopublishing company.Ltd., Seoul, Korea).

The present invention has been made in accordance with the above requirements, the present invention is derived from the pelargonium sp. (Pelargonium sp.) Material to induce and proliferate the cells in a specific cell growth medium to determine appropriate incubation conditions therefrom To establish the basis of the mass production system, from which the plant of Pelargonium sp. Nerve root To induce and multiply to prepare a framework for producing useful components.

In order to solve the above problems, the present invention provides a method for sterilizing and inoculating Pelargonium sp. Plants to induce and proliferate cells in a specific cell proliferation medium.

The present invention also provides pelargonium genus plant cells produced by the above method.

The present invention also provides a method of inducing and propagating adventitious roots into said cells.

The present invention also provides pelargonium genus plant root produced by the above method.

The present invention also relates to the genus Pelargonium sp. Comprising 3/4 MS medium to which NH ₄ ⁺ : NO ₃ ⁻ = 15: 30, sucrose 30 g · L ⁻¹ and NAA 5.0 mg · L ⁻¹ were added. ) plant Provided is a cell induction medium.

By inducing and proliferating cells from the method of propagating plant cells of the genus Pelargonium of the present invention, the optimum incubation conditions were established, thereby establishing the basis of the cell mass production system, and from these cells, the genus Pelargonium sp .) plant Nerve root Induction and propagation provided a framework for producing useful components. Therefore, if the cell culture method of the present invention and the root muscle prepared by the method is used, its useful value will be high as a plant tissue culture strategy for producing useful components.

In order to achieve the object of the present invention, the present invention relates to a method for inducing and propagating cells in a specific cell growth medium by sterilizing and inoculating Pelargonium sp. Plants, preferably cell growth medium. Is 3/4 MS medium to which NH ₄ ⁺ : NO ₃ ⁻ = 15: 30, sucrose 30 g · L ⁻¹ and NAA 5.0 mg · L ⁻¹ were added.

Pelargonium sp. Plants of the invention are preferably Pelargonium sidoides , but are not limited thereto.

The method of inducing the cells (callus) may be performed by a method commonly used in the art.

The present invention also provides plant cells of the genus Pelargonium produced by the above method. The cells of Pelargonium sidoides refer to cells which have been cultured by suspension culture of induced callus.

The present invention also provides a method of inducing and proliferating the adventitious muscle into the cells. The method of deriving the root canal from cells (calus) may be performed by methods commonly used in the art.

The present invention also provides pelargonium genus plant root produced by the above method.

The present invention also relates to the genus Pelargonium sp. Comprising 3/4 MS medium to which NH ₄ ⁺ : NO ₃ ⁻ = 15: 30, sucrose 30 g · L ⁻¹ and NAA 5.0 mg · L ⁻¹ were added. ) plant Provided is a cell induction medium.

The medium is preferably used for induction of pelargonium sidoides plant cells. It will be readily appreciated by those skilled in the art that the amount of ingredient added to the medium may vary within a range capable of achieving the effects of the present invention.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example 1 Induction and Proliferation of Cells and Abdominal Muscles

1.1 Cell and Abdominal Muscle Induction

In order to induce the cells (callus ) of Pelargonium sidoide, the young leaves of in-flight cultured plants were cut into 0.5 cmⅹ0.5 cm and 5 mg / L of NAA (α-naphthalene acetic acid) and 0.1 mg / of kinetin. L, sucrose 30 g / L, MS medium (Murashige T and Skoog F (1962) Physiol Plant 15 (3): 473-497) with addition of 2.3 g / L zeolite was inoculated. In addition, in order to induce the root of Pelargonium sidoides , young leaves of incubated plants were cut into 0.5 cm ⅹ 0.5 cm, 1 mg / L IBA (indolebutyric acid), 30 g / L sucrose, 2.3 g / L 3/4 MS medium (Wu, CHHN Murthy, EJ Hahn, KY Paek (2007) Biotech. Lett. 29: 1179-1182) added. The medium was adjusted to pH 5.8 using 0.1 N NaOH, sterilized using autoclave at 121 ° C. for 25 minutes, and then 30 mL each was dispensed into plastic Petri dishes (9.0φ × 1.0 h cm). Each culture was carried out for 8 weeks at 25 ± 2 ℃ dark conditions.

1.2. Cell and Abdominal Muscle Proliferation

Induced pelargonium sidoides cells and adventitious roots were transferred to the same medium, respectively, and passaged at 3 week intervals to proliferate. The culture was performed at 25 ± 2 ° C. dark conditions.

Example 2 Effect of Growth Regulators on the Growth of Cells and Abdominal Muscles

2.1. Effect of Auxin on Growth of Cells and Abdominal Muscle

To investigate the concentration of auxin suitable for the proliferation of pelargonium sidoides cells, NAA and IBA were added to MS medium containing 30 g / L sucrose and 2.3 g / L zeolite 1, 3, 5, 7, 9, respectively. IAA (indoleacetic acid), NAA, IBA in 3/4 MS medium containing sucrose 30 g / L and 2.3 g / L of zeolite, Were treated with 0.0, 0.05, 0.1, 0.5, 1, 2, 4 mg / L, respectively. The medium was dispensed in 30 mL each in a plastic Petri dish, and the culture was performed in 5 repeats per treatment for 3 weeks at 25 ± 2 ° C. dark conditions. After 3 weeks of cultivation, the ratio of dry weight, dry weight to dry weight, and growth rate were examined.

The dry weight was measured after drying for 24 hours in a dryer (FO-600M, jeio tech, Chengju, Korea) fixed at 60 ℃, and the dry weight ratio (% dry weight) to live weight was Kim (Kim, YS (2002) Panax ginseng CA Meyer Ginsenoside production through bioreactor culture of the root canal, PhD Thesis, Chungbuk National University, Cheongju, Korea) was calculated using the following equation.

Ratio of dry weight to live weight = dry weight / live weight ⅹ 100

Based on the initial inoculation amount, the method of Zhang et al. (Zhang, YH, JJ Zhong, and JT Yu (1996) J. Biotechnol. 51: 49-56) was used to determine the cell growth rate after 3 weeks of culture. According to the following equation.

Cell growth rate = [(amount of last grown cells)-(amount of initially seeded cells)] / (amount of initially seeded cells) / (growth period)

※ Growth period = Growth days (day)

Table 1. Pelargonium sidoides cell growth according to auxin type and concentration after 3 weeks of culture

As shown in FIG. 1 and Table 1, in the cell culture of Pelargonium sidoides , when 5 mg / L NAA was treated, the dry weight of the cells was 12.78 g and the% dry weight was 3.50 g. By obtaining it can be seen that the optimum concentration for cell culture. Compared with IBA, NAA treatment showed better cell growth, and especially the NAA 5mg / L concentration showed that the cell weight and dry weight of the cells were more than two times higher.

2.2. Effects of Cytokine on the Growth of Cells and Abdominal Muscles

In order to investigate the concentration of cytokinin suitable for the growth of Pelargonium sidoides cells, MS medium containing 5 mg / L NAA, 30 g / L sucrose and 2.3 g / L zeolite, which had the best cell growth in the auxin experiment, BA (benzyladenine) and kinetin concentrations were treated with 0.01, 0.05, 0.1, 0.3, 0.5 mg / L, respectively. Cytokinin concentration test suitable for the growth of Pelargonium sidoides root muscle.Sucrose 30 g / L in 3/4 MS medium supplemented with 0.1 mg / L of IBA and 0.1 mg / L of NAA, which showed the best growth of root muscle in auxin experiments. 2.3 g / L of gellite was added and the concentrations of BA and kinetin were treated with 0.01, 0.03, 0.05, 0.07, and 0.1 mg / L, respectively. The medium was dispensed 30 mL in a plastic Petri dish, and the culture conditions and irradiation items were the same as in Experiment 2.1.

Table 2. Pelargonium sidoides cell growth according to cytokinin type and concentration after 3 weeks of culture

As shown in FIG. 2 and Table 2, the cell growth in NAA 5mg / L alone treatment group was significantly higher in both live weight and dry weight than in the mixed treatment with cytokinin. Was found to be the optimal medium.

Example 3 Effect of Medium Composition on Growth of Cells and Abdominal Muscles

3.1. Effect of Inorganic Salt Concentration on the Growth of Cells and Abdominal Muscles

In order to investigate the concentration of inorganic salts suitable for growth of Pelargonium sidoides cells and abscess muscle, the total inorganic salt concentration of MS medium was adjusted to 1/4, 1/2, 3/4, 1, 2 times. Sucrose concentration in the medium was adjusted to 30 g / L, NAA 5 mg / L, IBA 0.1 mg / L was added respectively. Inoculation was performed by placing 70 mL of medium into a 250 mL Erlenmeyer flask, inoculating 50 g / L and 5 g / L of cells and adrenal muscle, and then suspending culture for 3 weeks in a dark condition at 20 ± 2 ° C. . After three weeks of culture, the ratio of dry weight, dry weight, dry weight to live weight, growth rate, and water potential of the medium were examined. Cells were measured in vivo after removing all the moisture in the cells using a vacuum pump (vacuum pump).

Table 3. Pelargonium sidoides cell growth according to MS medium concentration after 3 weeks of culture

As shown in FIG. 3 and Table 3, the growth of pelargonium cells was not significantly different in 3/4 MS medium and 1 MS medium, but in 3/4 MS medium, both live weight and dry weight were slightly higher and in long-term culture Considering the reduction of production cost and the stable maintenance of cultured cells, 3/4 medium was judged to be an appropriate level.

3.2. Effect of Nitrogen Sources on Growth of Cells and Abdominal Muscles

To investigate the effect of NH ₄ ⁺ : NO ₃ ^- ratio on Pelargonium sidoides cells and adventitious growth, the NH ₄ ⁺ : NO ₃ ^- ratio was set to 0:45, 7.5: 37.5, 15:30, 22.5: 22.5, 30:15, 37.5: 7.5, and 45: 0, respectively. NH ₄ NO ₃ and KNO ₃ were used to control the ratio of the two nitrogen sources, and all treatments were added with NAA 5 mg / L and sucrose 30 g / L in 3/4 MS medium except nitrogen source. In culture, 0.1 mg / L of IBA and 30 g / L of sucrose were added. The culture conditions were the same as in Experiment 3.1, and after 3 weeks of culture, the weight of dry weight, dry weight, dry weight to weight, growth rate, and amount of inorganic elements remaining in the medium were examined.

Inorganic ion content analysis in medium

After 3 weeks of culture, the medium was collected and the amount of NH ₄ ⁺ , NO ₃ ⁻ , P, K, Ca, and Mg remaining in the medium was measured. By diluting the culture medium in each treatment group with 10, 30, 60, 100 drain NH ₄ ^+, P, K, Ca, Mg was measured using an atomic absorption photometer (AA-6710F, Shimadzu, Kyoto , Japan), NO 3 ^- an HPLC (Waters 600S controller, Water 26 pump, Water Co., Milford, USA) IC-Pak HR column equipped with a (4.6ⅹ75mm, Waters Co., Mildfold, USA) 1.6 mM NaHCO 3 /1.2 mM Na 2 to CO ₃ was eluted at a flow rate of 1.0 mL / min as a mobile phase and detected by a conductivity detector (Yu KW (2000) PhD Thesis, Chungbuk National University, Cheongju, Korea). The content of P was measured using a molecular photometer (Uvikon-930, Kontron Instruments Co., Zurich, Switzerland) at a wavelength of 470 nm by vanadate method.

Table 4. Pelargonium sidoides cell growth according to NH ₄ ⁺ : NO ₃ ⁻ ratio after 3 weeks of culture

As shown in Figure 4 and Table 4, when the NH ₄ ⁺ : NO ₃ ^- ratio in the medium was adjusted to 15:30, it was confirmed that the ratio of nitrogen source was the most suitable for cell culture, showing the highest in vivo weight and dry weight.

       3.3. Effect of Glucose Concentration on Growth of Cells and Abdominal Muscle

The sucrose concentration in the medium was varied to 0, 1, 3, 5, 7, 9% to investigate the sugar concentration suitable for the growth of cells and root muscles. Other medium conditions were adjusted in the same manner as the previous experiment.

The culture conditions were the same as in Experiment 3.1, and after 3 weeks of culture, the weight ratio of dry weight, dry weight, dry weight to live weight, growth rate, water potential of medium, and amount of sugar remaining in the medium were examined.

Sugar analysis in medium

After 3 weeks of culture, the medium was collected, filtered through a 0.2 μm membrane filter, diluted 10-fold with distilled water, and 1 mL was used for analysis. Diluted media was equipped with a carbohydrate column (4.6 × 250 mm, Waters Co., Mildfold, USA) on HPLC (Waters 2690, Waters Co., Mildfold, USA) to add 80% acetonitrile to the mobile phase of 1.0. Moving to a flow rate of mL / min was detected by RI detector (Yu KW (2000) PhD Thesis, Chungbuk National University, Cheongju, Korea).

Table 5. Pelargonium sidoides cell growth according to sucrose concentration after 3 weeks of culture

As shown in FIG. 5 and Table 5, it was confirmed that the treatment group with the highest concentration of 3% sugar in vivo and dry weight was suitable for pelargonium cell culture.

Figure 1 is a result showing the comparison of Pelargonium sidoides cell growth according to auxin type and concentration after 3 weeks of culture,

Figure 2 is a result showing the comparison of Pelargonium sidoides cell growth according to the type and concentration of cytokinin after 3 weeks of culture,

Figure 3 is a result showing the comparison of Pelargonium sidoides cell growth according to MS medium concentration after 3 weeks culture,

Figure 4 is a result showing the comparison of Pelargonium sidoides cell growth according to the NH ₄ ⁺ : NO ₃ ^- ratio after 3 weeks of culture,

Figure 5 is a result showing the comparison of Pelargonium sidoides cell growth according to sucrose concentration after 3 weeks culture.

Claims (7)

In a method of inducing and propagating cells by sterilizing and inoculating Pelargonium sidoides plants, the cell proliferation medium is NH ₄ ⁺ : NO ₃ ⁻ = 15: 30, sucrose 30 g · L ⁻¹ and NAA ( α-naphthalene acetic acid) A method characterized by consisting of 3/4 MS medium to which 5.0 mg · L- ¹ is added. delete delete delete delete Pelargonium sidoides containing 3/4 MS medium with NH ₄ ⁺ : NO ₃ ⁻ = 15: 30, sucrose 30 g · L ⁻¹ and NAA 5.0 mg · L ⁻¹ Medium for inducing plant cells. delete

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