KR100476847B1 - Method of bioreactor culture of adventitious roots - Google Patents

Abstract

The present invention relates to a method for culturing irregular roots using a bioreactor, which sterilizes and inoculates seed plants to induce and proliferate callus; Inducing and propagating adventitious roots from callus; And culturing the root muscle to harvest the root muscle by culturing in the bioreactor, the method comprising: (1) supplying a gas having an oxygen concentration of 25 to 100% in the bioreactor; (2) supplying a gas having a carbon dioxide concentration of 0.5 to 20%; And (3) at least one step selected from the step of supplying a gas having an ethylene concentration of 5 to 100 ppm, when culturing ginseng, wild ginseng, or camphor ginseng using a bioreactor. By supplying oxygen, carbon dioxide, and / or ethylene gas at appropriate concentrations for a period of time, much higher yields can be obtained than with conventional culture processes, which is considered to be of value as a future plant tissue culture strategy.

Description

Cultivation method of root muscle using a bioreactor {METHOD OF BIOREACTOR CULTURE OF ADVENTITIOUS ROOTS}

The present invention relates to a method of culturing irregular roots using a bioreactor, which artificially regulates the composition of gas (O ₂ , CO ₂ , C ₂ H ₄ ) supplied into the bioreactor to increase the productivity of the root muscles and metabolize them. It is related to the method of culturing the roots of ginseng ( Panax ginseng CA Meyer), wild ginseng and camphor ginseng with increased product content.

Many reports have been reported in specialized journals on callus and intestinal root cultures derived from hairy roots and plant tissues in which ginseng, wild ginseng and camphor ginseng were transformed with A. rhizogenes . Most of these reports are related to the analysis of physical and chemical factors affecting hairy root and cell proliferation. Recently, many experimental results on ginseng root muscle cultivation have been published. In particular, cultivation of root muscle using a bioreactor has a big advantage that it can be mass-produced throughout the year and to produce stable secondary metabolites (Yu. KW, EJ Hahn, and KY Paek. 2000. Production of ginseng adventitious roots using bioreactors. Korean J. Plant Tiss. Cult. 27: 309-315.)

In case of arrhythmia cultivation using a bioreactor, the air supplied to the bioreactor until now is the same as the air composition in the general atmosphere, and is composed of 78% nitrogen, 20.8% oxygen, 0.9% argon, 0.03% carbon dioxide, neon, helium, etc. The productivity (live weight) of the root muscle by this air composition generally corresponds to 10-15% of the incubator volume.

The present invention provides a cultivation method of the root muscles to increase the productivity of the root muscles and increase the metabolite content by artificially controlling the composition of air supplied into the bioreactors when culturing ginseng, wild ginseng or camphor ginseng using a bioreactor The purpose is to do that.

Culture method of the root of the present invention for achieving the above object,

Sterilizing and inoculating the parent plants to induce and proliferate callus;

Inducing and propagating adventitious roots from callus; And

In the method of cultivating the root of the root muscle to harvest the root of the root muscle by culturing in the bioreactor,

In the bioreactor,

(1) supplying a gas having an oxygen concentration of 25-100%;

(2) supplying a gas having a carbon dioxide concentration of 0.5 to 20%; And

(3) at least one step selected from the step of supplying a gas having an ethylene concentration of 5 to 100 ppm.

Here, the parent plant may be at least one of ginseng, wild ginseng, and camphor ginseng, and the culture form of the plant includes the root and hair root, the cell.

The present invention is a gas (O ₂ , CO ₂ , C ₂ H ₄ ) component supplied to the bioreactor when incubating the root of ginseng ( Panax ginseng CA Meyer), wild ginseng or camphor ginseng using a bioreactor The purpose of this study is to investigate the effects on the growth and production of polysaccharides and metabolites, and to increase the metabolites content by increasing the productivity of the root muscle by artificially adjusting the composition of the gas supplied into the bioreactor.

According to the present invention, the effect of the supply of oxygen, carbon dioxide and ethylene gas into the bioreactor in the root muscle culture is estimated to be as follows.

1. Effect of Oxygen Supply on Abdominal Muscle Culture

In general, when the plant's respiratory substrate is glucose, oxygen stimulates the respiration of plant cells, producing more energy, resulting in increased plant growth.

C ₂ H ₁₂ O ₆ + 6O ₂ → 6CO ₂ + 6H ₂ O + energy

2. Effect of Carbon Dioxide Supply on Cultured Abdominal Muscle

Appropriate amounts of carbon dioxide are thought to increase the level of phosphoenol pyruvate carboxylase to promote carbon dioxide fixation and increase growth. Carbon dioxide, on the other hand, is thought to promote plant cell growth by enhancing the action of the Krebs cycle (Schnabl and Mayer, 1976, Planta. 131: 51-55). In addition, there have been reports of improved growth in various plant cells and tissue cultures (Schlatmanm et al. , 1994, Plant Cell Rep. 14: 157-160; Mirjalili and Linden, 1995, Biotechnol. Bioeng. 48: 173-187; and Zhong et al ., 2000, Enzyme Microb.Tech. 27: 714-723).

3. Effect of Ethylene Supply on Abdominal Muscle Culture

As a result of this experiment, when ginseng, wild ginseng or camphor ginseng roots absorb glucose and fructose in the medium, ethylene promotes their absorption. As a result, plants use a large amount of sugar and their growth increases.

The production process of the root muscle using the bioreactor according to the present invention consists of the following steps:

(1) selecting and sterilizing and inoculating the mother plant;

(2) induction and propagation of callus;

(3) induction and proliferation of the abscess;

(4) culturing in the bioreactor;

(5) supplying an appropriate amount of gas at an appropriate time during the incubation period;

(6) Harvest and composition analysis of wild ginseng roots.

1 is a process chart for the production of root muscle using bioreactors and gases.

Below, the production process of each of these steps is demonstrated concretely.

(1) Sterilization and inoculation by selecting a model plant

Ginseng, wild ginseng, or camphor ginseng was selected as a plant to sterilize root slices (2-3 mm2), and as growth regulators, IBA (indole butyric acid), NAA (1-naphthaleneacetic acid), 2,4-D ((2 Inoculate with MS (Murashige-Skoog), SH (Schenk and Hildebrandt), B5 (Gamborg), White medium with, 4-dichlorophenoxy) acetic acid).

(2) induction and proliferation of callus

After inoculation of the seedlings, it is incubated for 3 to 4 weeks in a cancerous state to induce callus in the root section. The callus thus induced is propagated by subcultured at intervals of 2 to 4 weeks in an optimal callus culture medium, that is, MS medium to which 1 mg / L 2,4-D is added.

(3) induction and proliferation of abscess muscle

The multiplied callus is transferred to MS, SH, B5 medium supplemented with IBA or NAA, and cultured for about 1 month to induce the root muscle.

(4) cultivation in a bioreactor

Among the induced root muscles, individuals with good growth are selected and transferred to a bioreactor for cultivation. The medium in which the growth of the negative root is good has a hydrogen ion concentration (pH) of 5.5 to 6.0 and a sugar concentration of 3 to 5%. The strength of the air supplied into the bioreactor is adjusted to 0.1 vvm and cultured for 30 to 50 days.

(5) supplying a suitable amount of gas during the incubation period

Various experiments are conducted to determine the most effective gas concentration for growth and metabolite content of the root canal, and the optimal gas concentration is selected. The concentration of each gas entering the bioreactor can be analyzed via gas chromatography (HP 6890, Hewlett Packard, USA).

(6) Harvest and composition analysis of the root muscle

a. Harvesting the roots of negative roots: The roots of ginseng, wild ginseng or camphor ginseng are cultured in a bioreactor for 30 to 50 days before harvesting the roots of negative roots. In the dry roots of the roots, the moisture is measured after completely removing moisture from the dryer (FO-600M, Jeio Tech Co., Korea) adjusted to 60 ℃.

b. Saponin Analysis: Extraction and crystallization of saponins were carried out by Yoshikaya and Furuya, 1987, Plant Cell Rep. 6: 449-453 and Williams A. et al. , 1996, J. Chromato. 775: 11-. It is carried out according to the method of 17). That is, 1 g of dried sarcophagus is refluxed twice in 50 ml of 80% ethanol for 1 hour, and the extract is filtered through filter paper (Advantec, Toyo, Japan) and separated from the solids. The ethanol extract is concentrated to 10 ml and dissolved in 50 ml of distilled water again, the aqueous layer is washed twice with the same volume of ether and extracted three times with water saturated butanol. The extract was washed twice with 20 ml of water, dried over a vacuum condenser, dissolved in 5 ml of methanol and filtered through a 0.45 μm PTEF (Gelman, USA) filter. Saponin content was measured using an Altec Platinum C18 or Noba-Pak C18 column (300 A, 5 μm) using HPLC with water and acetonitrile (Waters 2690 separation module; Waters 996 photodiode array detector; Waters millenium 2010 chromatography manager). Analyze In other words, the density gradient of water (A) and acetonitrile (B) starts at 18% (B) at the beginning, 18 to 23% (B) for 0 to 15 minutes, and 23 to 31% (for 15 to 18 minutes). B), 31-34% (B) at 18-33 minutes, and then increase the acetonitrile ratio for 7 minutes, then perform column washing and analyze continuously. The flow rate of the mobile phase is 0.6-1.0 ml / min, and the chromatogram is obtained at 203 nm. Wako (Japan) was used as the standard material and the saponin content was shown by adding the content of each component peak.

c. Polysaccharide Analysis: Suspend 0.2 g of dried sarcophagus by adding 5 ml of 5% (v / v) sulfuric acid (H ₂ SO ₄ ). The suspension is boiled in a water bath for 4 hours, acid hydrolyzed and centrifuged to give 0.2 mL of the filtrate, which is added to 6 mL of concentrated sulfuric acid and boiled in a water bath for 20 minutes. After cooling, 0.2 ml of a solution of carbazole-absolute ethanol (0-15%, v / v) was added, followed by color development in the dark for 2 hours, and then the absorbance was measured at 530 nm with a spectrophotometer. Obtain the content using galacturonic acid as a standard.

Hereinafter, specific features of the present invention will be described in detail with reference to the following examples. However, these Examples are only illustrative of the present invention, and the scope of the present invention is not limited to these.

Example

The wild ginseng root slices (2-3 mm2) were soaked in 70% ethanol for about 30 seconds, then sterilized with 2% sodium hypochlorite for about 25 minutes, sterilized by washing three times or more with sterile water, and then grown. Inoculation was carried out in MS medium to which 1 mg / L 2,4-D was added as a modulator and incubated for 3 to 4 weeks in a cancer state to induce callus, which was passaged at intervals of 2 to 4 weeks in the same medium.

The multiplied callus was transferred to MS medium to which 5 mg / l IBA or 2 mg / l NAA was added and incubated for about 1 month to induce root muscles. Was carried out. The pH of the medium was 5.5-6.0, the sugar concentration was 3-5%, and the strength of air supplied into the bioreactor was adjusted to 0.1 vvm for 30 to 50 days.

As shown below, the roots were harvested after incubating the root muscles while supplying various kinds of gases into the bioreactor for several periods as follows. Dry matter weight of the root muscle was measured, and saponin and polysaccharide were analyzed according to the above method.

1. Effect of Oxygen Supply on Wild Ginseng Root Culture

In the culture of wild ginseng (ginseng, camphor ginseng) root muscle using a bioreactor, the air is generally the same as the air composition, but oxygen takes up 20.8% in the general atmosphere. The same air composition (20.8% oxygen) was supplied to a 5 L bioreactor to incubate wild ginseng roots for 50 days to obtain 45.55 g of dry matter.

On the other hand, by adjusting the oxygen concentration to 30% or 40% and cultured wild ginseng roots while continuously feeding for 50 days, the growth and metabolites were analyzed.

Figure 2 is a graph showing the growth and metabolite content change of wild ginseng root muscle with oxygen supply during the pre-culture period, Figure 2a shows the growth amount, Figure 2b saponin content, and Figure 2c shows the polysaccharide content. As shown here, 48.85 g and 52.11 g of dry weight were obtained when the oxygen concentration was adjusted to 30% or 40% and continuously fed for 50 days of culture. In addition, it was found that the saponin and polysaccharide content also increased when oxygen was added.

Subsequently, various experiments were conducted in which 40% oxygen was supplied only during a specific period to investigate the efficiency and economics of oxygen supply.

Figure 3 is a graph showing the growth and metabolite content change of wild ginseng root muscle with oxygen supply during a specific period of culture, Figure 3a shows the growth, Figure 3b saponin content, and Figure 3c shows the polysaccharide content. As shown here, 56.53 g of the highest growth was obtained when 10 to 50 days of incubation was obtained, rather than continuing to supply oxygen for 0 to 50 days before the incubation period.

2. Effect of CO2 Supply on Cultured Wild Ginseng Roots

In the general atmosphere, carbon dioxide occupies 0.03%, and 5 l of air, which is the same as the atmospheric composition, was supplied to the bioreactor to cultivate wild ginseng root, and 43.32 g of dry matter was obtained after 50 days of culture.

On the other hand, by artificially adjusting the carbon dioxide concentration to 2.5% or 5%, the growth and metabolites were analyzed after culturing wild ginseng roots while continuously feeding for 50 days.

Figure 4 is a graph showing the growth and metabolite content change of wild ginseng root muscle with carbon dioxide during the pre-incubation period, Figure 4a shows the growth, Figure 4b saponin content, and Figure 4c shows the polysaccharide content. As shown here, supplying carbon dioxide concentrations of 2.5% and 5% yielded 51.65 g and 50.1 g of dry matter, respectively.

Subsequently, various experiments were conducted in which 2.5% of carbon dioxide was supplied only during a specific period to investigate the efficiency and economic efficiency of the carbon dioxide supply.

Figure 5 is a graph showing the growth and metabolite content of wild ginseng root muscle with carbon dioxide supply during a specific period of culture, Figure 5a shows the growth, Figure 5b saponin content, and Figure 5c shows the polysaccharide content. As shown here, 61.76 g of the highest growth was obtained when 10 to 50 days of incubation was obtained, rather than continuously supplying carbon dioxide for 0 to 50 days before the incubation period.

3. Effect of Ethylene Supply on Cultured Wild Ginseng Roots

Ethylene content in the general atmosphere was 0 ppm, and 5 g of air having the same composition as that of the atmospheric composition was fed to the bioreactor for incubation of wild ginseng root for 50 days to obtain 45.47 g of dry matter.

On the other hand, ethylene concentration was artificially adjusted to 10 ppm or 20 ppm and cultured wild ginseng roots while continuously feeding for 50 days, and then analyzed for growth and metabolites.

Figure 6 is a graph showing the growth and metabolite content change of wild ginseng root muscle with ethylene supply during the pre-incubation period, Figure 6a shows the growth amount, Figure 6b saponin content, and Figure 6c polysaccharide content. As shown here, feeding ethylene at 10 ppm and 20 ppm yielded 57.9 g and 55.17 g of dry matter, respectively.

Subsequently, various experiments were conducted in which 10 ppm of ethylene was supplied only for a specific period of time to investigate the efficiency and economics of the ethylene supply.

Figure 7 is a graph showing the growth and metabolite content of wild ginseng root muscle with ethylene supply during a specific period of culture, Figure 7a shows the growth, Figure 7b saponin content, Figure 7c shows the polysaccharide content. As shown here, 70.94 g of the highest growth was obtained when ethylene was fed for 0-30 days of cultivation, rather than continuously fed for 0-50 days before the cultivation period.

4. Effect of Mixed Gas Supply in Cultured Wild Ginseng Roots

In this experiment, the effects of single treatment with oxygen, carbon dioxide, and ethylene, two gas treatments, and three gas treatments were investigated. At this time, the supplied gas was 40% oxygen, 2.5% carbon dioxide, and 10 ppm ethylene, and two or more combination treatments were also configured at the same gas concentration. Each gas supply period was 10 to 50 days for oxygen and carbon dioxide and 0 to 30 days for ethylene for 50 days of total culture. In other words, the concentration of gas used in this experiment and the supply period are the optimal results of the previous experiment.

8 is a graph showing the growth and metabolite content change of wild ginseng root muscle with mixed gas supply during a specific period of culture, Figure 8a shows the growth, Figure 8b saponin content, and Figure 7c shows the polysaccharide content. Where A is the control, B is O ₂ , C is CO ₂ , D is C ₂ H ₄ , E is O ₂ + CO ₂ , F is O ₂ + C ₂ H ₄ , G is CO ₂ + C ₂ H ₄ , H is the case of O ₂ + CO ₂ + C ₂ H ₄ . As shown in FIG. 8, 46.53 g of dry weight was obtained in the conventional culture process, and the highest growth amount, 72.51 g, was obtained when the three gases of oxygen, carbon dioxide, and ethylene were treated simultaneously. In addition, since each gas was supplied only for a certain period of time, not for the entire period of incubation, less gas was used than 50 days of continuous gas supply, and in this case, more production could be achieved. There are advantages.

The following Table 1 shows the growth of wild ginseng roots with nitrogen and oxygen gas supply.

Treatment (G) in building Existing Culture (Control) 39.3 Nitrogen Gas 5% 36.3 Nitrogen Gas 10% 26.0 Oxygen 25% 41.5 Oxygen 30% 42.6

5. Effect of ethephon treatment on wild ginseng root culture

When ethanol generating ethylene gas was treated with 10 ppm and 30 ppm in medium, cultivation of wild ginseng root muscle yielded 62.81 g and 49.41 g of dry weight, respectively. At this time, 43.82 g of dry weight was obtained by the existing culture process.

The following Table 2 shows the growth and metabolite content of wild ginseng root of Ginseng by ethephon treatment.

Live weight (g) (G) in building Saponin content Existing Culture (Control) 561.41 43.82 2.25 ± 0.34 Ethephon 10 ppm 651.30 62.81 0.99 ± 0.02 Ethephon 30 ppm 484.51 49.41 0.84 ± 0.02

In summary, the experimental results according to the above embodiment, when the growth of wild ginseng ( Panax ginseng CA Meyer) in the root muscle using a bioreactor, when the concentration and supply period of oxygen injected into the bioreactor were adjusted, Compared with the production by the control (control), the production increased by 14.4 to 21.1%. In addition, it was possible to increase the growth of 19.2 to 24.1% for carbon dioxide and 27.3 to 37.5% for ethylene. On the other hand, when a combination of three gases of oxygen, carbon dioxide and ethylene was treated for a certain period of time it was possible to obtain a growth rate of up to 55.8%.

In the saponin accumulation by wild ginseng root muscle, saponin synthesis of about 5.42 mg / g was obtained by 1.5-fold increase compared to the saponin content (3.6 mg / g) by the conventional culture step in the oxygen-incubated step. In the polysaccharide content, oxygen treatment was 12.2%, which was slightly increased compared to the polysaccharide content of 11.4% by conventional culture.

On the other hand, when treated with the ethylene produced the ethylene gas, the growth of the roots of the roots of the growth of 43.3% compared to the conventional culture process.

According to the present invention, by using a bioreactor to supply oxygen, carbon dioxide, and / or ethylene gas at an appropriate concentration for a certain period of time in culture of ginseng, wild ginseng, or camphor ginseng, a much higher yield can be obtained than a conventional culture process. It may be of value as a future plant tissue culture strategy.

1 is a process chart for the production of root muscle using a bioreactor and gas,

Figure 2 is a graph showing the growth and metabolite content of wild ginseng root muscle with oxygen supply during the pre-culture period,

Figure 3 is a graph showing the growth and metabolite content of wild ginseng root muscle with oxygen supply during a specific period of culture,

4 is a graph showing the growth and metabolite content of wild ginseng roots with carbon dioxide during the pre-culture period,

5 is a graph showing the growth and metabolite content of wild ginseng roots with carbon dioxide supply during a specific period of culture,

6 is a graph showing the growth and metabolite content of wild ginseng roots with ethylene supply during the pre-culture period,

7 is a graph showing the growth and metabolite content of wild ginseng root muscle with ethylene supply during a specific period of culture.

8 is a graph showing the growth and metabolite content changes of wild ginseng root muscle with mixed gas supply during a specific period of culture.

Claims (7)

Sterilizing and inoculating the parent plants to induce and proliferate callus; Inducing and propagating adventitious roots from callus; And In the method of cultivating the root of the root muscle to harvest the root of the root muscle by culturing in the bioreactor, In the bioreactor, (1) supplying a gas having an oxygen concentration of 25-100%; (2) supplying a gas having a carbon dioxide concentration of 0.5 to 20%; And (3) at least one step selected from the step of supplying a gas of 5 to 100 ppm ethylene concentration. The method of claim 1, wherein the parent plant is selected from ginseng, wild ginseng and camphor ginseng. The method of claim 1, further comprising the step of treating the medium to contain 1 to 100 ppm of ethephon. The method of claim 1 wherein the gas is supplied for the entire incubation period. The method of claim 1, wherein at least one of a gas having an oxygen concentration of 25 to 100% and a gas having a carbon dioxide concentration of 0.5 to 20% is supplied for 10 to 50 days of culture. The method according to claim 1, wherein a gas having an ethylene concentration of 5 to 100 ppm is supplied for 30 days from the start of the culture. The method according to claim 1, wherein a gas having an oxygen concentration of 25 to 100%, a gas having a carbon dioxide concentration of 0.5 to 20%, and a gas having an ethylene concentration of 5 to 100 ppm are supplied together.