WO2008100016A1 - Method of bioreactor culture of echinacea purpurea adventitious roots - Google Patents

Method of bioreactor culture of echinacea purpurea adventitious roots Download PDF

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Publication number
WO2008100016A1
WO2008100016A1 PCT/KR2007/007021 KR2007007021W WO2008100016A1 WO 2008100016 A1 WO2008100016 A1 WO 2008100016A1 KR 2007007021 W KR2007007021 W KR 2007007021W WO 2008100016 A1 WO2008100016 A1 WO 2008100016A1
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adventitious root
culture
content
adventitious
growth
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PCT/KR2007/007021
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French (fr)
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Kee Yoeup Paek
Chun Hua Wu
Eun Joo Hahn
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Cbnbiotech Co., Ltd
Chungbuk National University Industry-Academic Cooperation Foundation
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Publication of WO2008100016A1 publication Critical patent/WO2008100016A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G22/00—Cultivation of specific crops or plants not otherwise provided for
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G7/00—Botany in general
    • A01G7/04—Electric or magnetic or acoustic treatment of plants for promoting growth
    • A01G7/045—Electric or magnetic or acoustic treatment of plants for promoting growth with electric lighting
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C12N5/0025—Culture media for plant cell or plant tissue culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/34—Sugars

Definitions

  • the present invention relates to a method of culture of the Echinacea purpurea adventitious root, including inducing and multiplying the adventitious root, elucidating an optimum in vitro culture condition therefrom, establishing the mass-production system of the adventitious root at large scale in bioreactor culture, and producing effective components from the adventitious root.
  • Echinacea is a medicinal plant originated from North America, which has been used by natives for curing of wound, burning, snake or insect bite, cold, infection, and inflammation. As a perennial herb, it belongs to Compositae family and includes many varieties. The representative kinds are Echinacea purpurea (E. purpurea), E. an- gustifolia, E. pallida, each has its own characteristic main chemical component. Echinacea is used in the form of an extract, tea, a tonic agent, a tablet, or an ointment. It has the effect best known as a nonspecific immunostimulating agent against respiratory infection, urinary tract infection, cancer, skin disease, and other many inflammatory processes, and it increases macrophage population and is known to kill cancer cells.
  • E. purpurea E. an- gustifolia
  • E. pallida each has its own characteristic main chemical component.
  • Echinacea is used in the form of an extract, tea, a tonic agent, a tablet, or an oin
  • the purpose of the present invention is to induce and multiply the adventitious root from the Echinacea purpurea, to elucidate an optimum in vitro culture condition therefrom, to establish the mass-production system of the adventitious root at large scale in bioreactor culture, and to produce effective components from the adventitious root.
  • the Echinacea purpurea was used as a plant material for inducing and multiplying the adventitious root.
  • an optimum in vitro culture condition was elucidated therefrom, mass-production system of the adventitious root at large scale in bioreactor culture was established, and an effective component from the adventitious root was produced.
  • a composition for culture media which can affect the growth of the adventitious root was determined, and an optimum condition for growing the roots in a bioreactor was also identified.
  • a method for improving the productivity by using a physical and chemical elicitor was provided.
  • the object of the present invention is to provide a method of culturing adventitious root comprising the steps of:
  • inoculum size of the adventitious root is 5 ⁇ 10g/L
  • [14] (4) culture period is from 3 to 5 weeks, and [15] (5) culture temperature is between 15-25 0 C.
  • the culture method of the present invention is an optimum method for culturing in a bioreactor by which selection of a genetically favored plant and multiplication in a relatively short period of time are possible.
  • the culture method of the present invention is highly valuable in that it can be strategically used for culturing plant tissues to obtain effective components therefrom.
  • Figure 1 includes pictures showing Echinacea purpurea adventitious roots which have been cultured in petri-dish, Echinacea purpurea adventitious root being cultured in 5L, 2OL, 500L, and IOOOL bioreactor, and the adventitious roots harvested therefrom.
  • Figure 2 is a graph showing the effect of temperature on the antioxidant enzyme activity in adventitious root culture
  • Figure 3 includes, for culturing adventitious root, a) a graph showing the effect of
  • Figure 4 is a graph showing the polyploidy of chromosomes between the Echinacea adventitious root and its leaf
  • FIG. 5 is a picture showing chromosome number of the Echinacea adventitious root
  • Figure 6 is an HPLC chromatogram of caffeic acid from Echinacea species, specifically, (a) E.palida cultivated root;b) E. angustiforlia cultivated root; c) E. purpurea cultivated root; d) E. purpurea adventitious root,
  • Figure 7 is an HPLC chromatogram of standard caffeic acid derivatives for analyzing chemical compounds comprised in Echinacea purpurea adventitious roots.
  • the present invention relates to a method of bioreactor culture of Echinacea purpurea adventitious roots. Specifically, the adventitious root was induced and multiplied, an optimum culture condition was elucidated therefrom, mass-production system of the adventitious root at large scale in bioreactor culture was established, and an effective component from the adventitious root was produced. To do so, a composition for culture media which can affect the growth of the adventitious root was determined, and an optimum condition for growing the roots in a bioreactor and a content of the effective component was also identified. Moreover, in order to increase a content of polyphenolic compounds and caffeic acid, which are secondary metabolites of the produced adventitious root, a method for improving the productivity by using a physical and chemical elicitor was provided.
  • the object of the present invention is to provide a method of culturing adventitious root comprising the steps of:
  • the inoculun size of the adventitious root is 7g/L
  • air supply amount is 0.1 vvm
  • culture period is 5 weeks
  • culture temperature is 2O 0 C.
  • the present invention further includes the culture method of the adventitious root which includes l/2mMS culture medium is further added 2 weeks after the start of the culture, in order to improve the growth of the adventitious root.
  • the present invention further includes the culture method of the adventitious root, which includes that from the initial point of the growth the adventitious roots are treated everyday in a growth chamber having a controlled light intensity of 30 ⁇ molm ⁇ 2 s "1 with an illumination cycle of 3h/21h (light/dark) and then cultured, in order to increase the content of the polyphenolic compound, which is a secondary metabolite of the adventitious root.
  • the present invention further includes the culture method of the adventitious root, which includes culturing under UV-A fluorescent lamp (2OW, Sankyo Denki, Japan) as a physical elicitor, placed 50cm above the bioreactor, everyday for Ih for one week from the fourth week of the culture, in order to increase the content of the polyphenolic compound which is a secondary metabolite of the adventitious root.
  • the culture method of the adventitious root includes culturing under UV-A fluorescent lamp (2OW, Sankyo Denki, Japan) as a physical elicitor, placed 50cm above the bioreactor, everyday for Ih for one week from the fourth week of the culture, in order to increase the content of the polyphenolic compound which is a secondary metabolite of the adventitious root.
  • the present invention further includes the culture method of the adventitious root, which includes treating the roots with nitrogen monoxide at 100 ⁇ mol as a chemical elicitor at the fourth week of the culture and then culturing them, in order to increase the content of the polyphenolic compound which is a secondary metabolite of the adventitious root.
  • the present invention further includes the culture method of the adventitious root, which includes treating the roots with salicylic acid at 200 ⁇ mol as a chemical elicitor at the fourth week of the culture, in order to increase the content of the polyphenolic compound which is a secondary metabolite of the adventitious root.
  • the present invention further includes the culture method of the adventitious root, characterized in that the plant is selected from ginseng, wild ginseng or cultivated wild ginseng.
  • the present invention further includes the culture method of the adventitious root, characterized in that the plant is Echinacea purpurea.
  • the present invention further includes the culture method of the adventitious root, characterized in that the adventitious root cultured at large scale is genetically stable as being identical with the parent in terms of the chromosome polyploidy and its number.
  • the present invention further provides adventitious root that is cultured by the culture method of the present invention.
  • Example 1 The induction of the callus and the adventitious root
  • the Echinacea purpurea used in the present invention was seedling which had been grown from the germinated seed purchased from Canada. After cutting a stem and a leaf of the plant with an appropriate size for the surface sterilization before inoculation, they were immersed in 70% ethanol for 30 second, mixed with 4% sodium hypochlorite for 5 minutes, sterilized, washed with sterile water, and finally made in small pieces (lx ⁇ .5x ⁇ .3cm) which were then used as a culture material.
  • MS Murashinge and Skoog, 1962
  • Basal medium containing the naphthalene acetic acid (NAA) 2mg/L, the growth regulator of the benzyladenine (BA) 0. lmg/L, sucrose of 30g/L, and the gelite of 2.0g/L were inoculated and cultured for 8 weeks in a dark incubation room at 22+1 0 C.
  • sucrose of 30g/L, the gelite of 2.0g/L, and the NAA of 0.5mg/L as a growth regulator were added to the MS basal medium and then incubated in the dark incubation room at 22+1 0 C for 8 weeks.
  • Each of the culture medium was adjusted to pH 5.8 by using IN NaOH, sterilized for 20 minutes using autoclave at 121 0 C, and then aliquoted to a plastic cell culture dish 30m# each.
  • the culture medium of 4L was added in the airlift type BTBB of the total volume 5L and incubation was performed for 5 weeks at 22+1 0 C in dark condition. Each treatment was carried out in duplicate.
  • the effects of the maximn concentration, the inoculum size of the adventitious root, the air supply amount, and the culture period were investigated with respect to the growth of the adventitious root and the content of phenol, flavonoid, and caffeic acid derivatives, respectively.
  • the content of phenol, flavonoid, and caffeine acid derivative etc. was analyzed using an extract of the dried plant body obtained by using 80% methanol.
  • the phenol content was measured by 760nm absorbance of Folin-Ciocalteu reagent against gallic acid standards.
  • the flavonoid was measured with an absorbance at 510nm using catechin standards.
  • the HPLC analysis condition for measuring the content of the caffeic acid derivatives includes the use of water and acetonitrile as a mobile phase and the XTerra
  • the 1/2 MS treatment is the most effective as the fresh weight (e.g., weight of raw and fresh root) 73.6g/L, the dry weight 10.03g/L, the total phenol content 61.14mg/g DW, the total flavonoid content 38.30mg/g DW, and total caffeic acid derivative content 38.28mg/g DW. Therefore, the concentration of the inorganic materials required for the culture maximn for the growth of the Echinacea purpurea adventitious root was found to be 1/2MS (table 1 and table X).
  • Echinacea purpurea adventitious root the air supply amount more than 0.1 vvm inhibited the growth of the adventitious root and content of a secondary metabolite.
  • the fresh weight and dry weight, and the amount of secondary metabolites increased a little bit for the group to which the air supply was slowly increased up to 0.3 vvm.
  • the air supply amount suitable for the growth of the Echinacea purpurea adventitious root was found to be 0.1 vvm, with the fresh weight 70.1g/L, the dry weight 9.06g/L, the total caffeic acid derivatives content 37.39mg/g DW (table 5 and the table 6).
  • the inoculum size of adventitious root was 7.OgL "1 and the air supply amount was 0.1 vvm.
  • the composition of supplied air was the same as atmospheric air (N 2 78%, O 2 20.8%, CO 2 0.03%, Ne, He) and the total culture period was 8 weeks.
  • the content of phenolic compound and caffeic acid derivatives, pH of the medium, EC(electrical conductivity), and the content of sugar, cation and anion were analyzed with the interval of one week.
  • the culture condition was the same as the above Example 2-1.
  • the appropriate culture period of the Echinacea adventitious root is found to be five weeks in 5L bioreactor.
  • the content of sucrose in the culture medium drastically dropped from the beginning of the culture to week 2, suggesting that it was degraded to glucose and fructose and then used by the plant.
  • the content of cation and anion in the culture mediun drastically dropped from week 2 and then exhausted, causing an imbalance of the culture medium mesotrophy.
  • Example 3 Effectsofadditionofthemediumhavingdifferentconcentration. different culturetemperatureandillumination cvcleonthegrowthof adventitious rootinbioreactor
  • CTBB having total volume of 5L
  • the effect of the addition of the maximn having different concentration, different culture temperature and illumination cycle on the growth of adventitious root in bioreactor on the growth of the adventitious roots, and the content of phenolic compounds, flavonoid, and caffeic acid derivatives were determined.
  • the culture medium having different concentration was added in week 2 and week 3, in order to increase the growth of the adventitious root and content of a secondary metabolite.
  • the fresh weight and dry weight were higher for the group to which the culture medium was further added compared to the control group.
  • the content of a secondary metabolite showed a level equivalent to the control group, while it is lower for the group to which a higher amount of the culture medium was added compared to the control group.
  • the treatment group showing excellent fresh weight, dry weight, and total secondary metabolite content was 1/2MS culture maximn group wherein the 1/2 MS medium was added at week 2, with the fresh weight 83.1g/L (1.27 times), the dry weight 14.76g/L (1.5 times), the total phenol content 59.21mg/g DW, the total flavonoid 34.19mg/g DW, and the total caffeic acid derivative content 36.15mg/g DW which are the equivalent content with the control (table 7 and table 8).
  • the activity of SOD was measured by reading an absorbance at 560nm after irradiating with a fluorescent lamp the crude enzyme solution in a transparent tube, and then the measured absorbance was compared to that of the control.
  • the G-POD activity was measured by following the decrease of an absorbance at 470nm in accordance with the oxidation of a guaiacol under the presence of H 2 O 2 .
  • the activity of the CAT (catalase) was measured at 240nm by following the concentration reduction of H 2 O 2 when the enzyme extract of predetermined amount was added to the reaction solution comprising H 2 O 2 .
  • the activity of the APX was measured by the absorbance decrease at 290nm in accordance with the oxidation of the ascorbic acid as H 2 O 2 was added to the crude enzyme solution.
  • the radical eliminating ability was measured by using DPPH method. Specifically, after DPPH being added to the methanol extract of the dry adventitious root and keeping the mixture for 10 minute, the absorbance decrease at 520nm was measured.
  • IBA 2.0mg/L] culture medium was added to 3L bioreactor and the adventitious root of Echinacea was cultured for five weeks at 2O 0 C under dark condition.
  • the roots were irradiated every day for 0h,0.5h,lh,4h, respectively by using an UV-A fluorescent lamp (2OW, Sankyo Denki, Japan) during week 4, and the lamp was placed 50 an above the bioreactor. The experiment was carried out in duplicate.
  • the fresh weight, dry weight, total phenol content, total flavonoid content, and total caffeic acid derivative content were all increased compared to the control group.
  • Ih treatment group was the best, with the fresh weight of 67.7g/L, dry weight of 10.59g/L, total phenol content of 61.72r ⁇ g/g DW(1.20 times), total flavonoids content of 36.52mg/g DW(1.14 times), and total caffeic acid content of 42.38mg/g DW(1.24 times). (Table 13, Table 14).
  • Echinacea species that were used in the present analysis had been purchased from Canada and they were three-year-old cultivated product.
  • Echinacea purpurea adventitious root was obtained from the culture in 2OL bioreactor by using an optimal culture medium.
  • HPLC analysis condition includes the use of water and acetonitrile as a mobile phase and the XTerra
  • echinacoside was the only one detected of which content was 6.70mg/g DW.
  • caftaric acid 0.34mg/g DW, echinacoside 5.96mg/g DW, cichoric acid 0.66mg/g DW, total 6.97mg/g DW were detected.
  • caftaric acid 2.39mg/g DW, cichoric acid 6.18mg/g DW, total 8.57mg/g DW were detected.
  • E.purpuread caftaric acid 2.39mg/g DW, cichoric acid 6.18mg/g DW, total 8.57mg/g DW were detected.
  • the Echinacea adventitious root was harvested with the fresh weight 1.07Kg, the dry weight 0.14Kg, and the total caffeic acid derivative content 36.68mg /g DW.
  • the Echinacea adventitious root was harvested with the fresh weight 26.80kg, the dry weight 3.62Kg, and the total caffeic acid derivative content 26.84mg/g DW.
  • the adventitious root was grown to the fresh weight 41.02Kg, the dry weight 5.10kg, and the caffeic acid derivative content 32.03mg/g DW.

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Abstract

The present invention relates to a method of culture of the Echinacea purpurea adventitious root, including induction and multiplication of the adventitious root, elucidation of an optimum in vitro culture condition therefrom, establishment of the mass-production system of the adventitious root at large scale in a bioreactor, and production of effective components from the adventitious root.

Description

Description
METHOD OF BIOREACTOR CULTURE OF ECHINACEA PURPUREA ADVENTITIOUS ROOTS
Technical Field
[1] The present invention relates to a method of culture of the Echinacea purpurea adventitious root, including inducing and multiplying the adventitious root, elucidating an optimum in vitro culture condition therefrom, establishing the mass-production system of the adventitious root at large scale in bioreactor culture, and producing effective components from the adventitious root. Background Art
[2] Echinacea is a medicinal plant originated from North America, which has been used by natives for curing of wound, burning, snake or insect bite, cold, infection, and inflammation. As a perennial herb, it belongs to Compositae family and includes many varieties. The representative kinds are Echinacea purpurea (E. purpurea), E. an- gustifolia, E. pallida, each has its own characteristic main chemical component. Echinacea is used in the form of an extract, tea, a tonic agent, a tablet, or an ointment. It has the effect best known as a nonspecific immunostimulating agent against respiratory infection, urinary tract infection, cancer, skin disease, and other many inflammatory processes, and it increases macrophage population and is known to kill cancer cells.
[3] There has been a report suggesting that immunostimulating function of Echinacea is due to a lipophilic and polarity fraction of the extract comprising alkamide, caffeine acid derivative (cichoric acid), glycoprotein, and polysaccharide. The lipophilic fraction such as alkamide mainly contributes to the immunostimulation by the Echinacea alcoholic extract, and there is a report that the alkamide fraction promotes the phagocytosis both in vivo and in vitro. It has been reported that as an antiinflammatory agent alkamide of Echinacea, which is highly unsaturated carboxylic acid isobutylamide having olefinic or acetylene linkage, inhibits a cyclooxygenase and 5-lipoxygenase. It is also reported in many research works that the echinacoside (echinacoside, disaccharide) and the echinacin (polysaccharide), both obtained from roots of echinacea, have an effect of controlling an immune system by providing an antibacterial activity and antifungal activity, etc. For example, in Korean Patent No. 10-0497152 Bl, ethyl acetate fraction of the methanol extract of the Echinacea an- gustifolia root, which has an anticancer activity against blood cancer cell, has been disclosed.
Disclosure of Invention
Technical Problem
[4] It is difficult to satisfy the demand for Echinacea only by harvesting wild plants, and therefore a more efficient way of producing it is required. The mass production by the tissue culture is now widely used for many kinds of plants since it can be used to select an individual plant that is genetically favored and can allow its multiplication in a relatively short period of time. Thus, the purpose of the present invention is to induce and multiply the adventitious root from the Echinacea purpurea, to elucidate an optimum in vitro culture condition therefrom, to establish the mass-production system of the adventitious root at large scale in bioreactor culture, and to produce effective components from the adventitious root. Technical Solution
[5] Thus, in the present invention, the Echinacea purpurea was used as a plant material for inducing and multiplying the adventitious root. In addition, an optimum in vitro culture condition was elucidated therefrom, mass-production system of the adventitious root at large scale in bioreactor culture was established, and an effective component from the adventitious root was produced. To do so, a composition for culture media which can affect the growth of the adventitious root was determined, and an optimum condition for growing the roots in a bioreactor was also identified. Moreover, in order to increase a content of polyphenolic compounds, which is a secondary metabolite of the produced adventitious root, a method for improving the productivity by using a physical and chemical elicitor was provided.
[6] The object of the present invention is to provide a method of culturing adventitious root comprising the steps of:
[7] - steriliang and inoculating a plant to induce and to multiply callus;
[8] - inducing and multiplying the adventitious root from the callus; and
[9] - culturing the adventitious root in a bioreactor and harvesting it,
[10] characterized in that,
[11] (1) l/2mMS culture mediun [modified MS medium 1/2 MS (NH 4 +:NO3 =5:25) + sucrose 50g/L + IBA 2.0mg/L] is used as a culture medium,
[12] (2) inoculum size of the adventitious root is 5~10g/L,
[13] (3) air supply amount is 0.05~0.3vvm,
[14] (4) culture period is from 3 to 5 weeks, and [15] (5) culture temperature is between 15-250C.
[16] It is still another object of the present invention to provide adventitious root that is cultured by the culture method of the present invention.
Advantageous Effects
[17] As clearly shown in the examples of the present invention, induction and multiplication of the adventitious root, elucidation of an optimun in vitro culture condition therefrom, establishment of the mass-production system of the adventitious root at large scale in bioreactor culture are possible according to the culture method of the Echinacea purpurea adventitious root of the present invention. Furthermore, the culture method of the present invention is an optimum method for culturing in a bioreactor by which selection of a genetically favored plant and multiplication in a relatively short period of time are possible. Thus, the culture method of the present invention is highly valuable in that it can be strategically used for culturing plant tissues to obtain effective components therefrom. Brief Description of the Drawings
[18] Figure 1 includes pictures showing Echinacea purpurea adventitious roots which have been cultured in petri-dish, Echinacea purpurea adventitious root being cultured in 5L, 2OL, 500L, and IOOOL bioreactor, and the adventitious roots harvested therefrom.
[19] Figure 2 is a graph showing the effect of temperature on the antioxidant enzyme activity in adventitious root culture,
[20] Figure 3 includes, for culturing adventitious root, a) a graph showing the effect of
NO on SOD activity, b) a graph showing the effect of NO on G-POD activity, and c) a graph showing the effect of NO on APX activity,
[21] Figure 4 is a graph showing the polyploidy of chromosomes between the Echinacea adventitious root and its leaf,
[22] Figure 5 is a picture showing chromosome number of the Echinacea adventitious root,
[23] Figure 6 is an HPLC chromatogram of caffeic acid from Echinacea species, specifically, (a) E.palida cultivated root;b) E. angustiforlia cultivated root; c) E. purpurea cultivated root; d) E. purpurea adventitious root,
[24] Figure 7 is an HPLC chromatogram of standard caffeic acid derivatives for analyzing chemical compounds comprised in Echinacea purpurea adventitious roots. Mode for the Invention [25] The present invention relates to a method of bioreactor culture of Echinacea purpurea adventitious roots. Specifically, the adventitious root was induced and multiplied, an optimum culture condition was elucidated therefrom, mass-production system of the adventitious root at large scale in bioreactor culture was established, and an effective component from the adventitious root was produced. To do so, a composition for culture media which can affect the growth of the adventitious root was determined, and an optimum condition for growing the roots in a bioreactor and a content of the effective component was also identified. Moreover, in order to increase a content of polyphenolic compounds and caffeic acid, which are secondary metabolites of the produced adventitious root, a method for improving the productivity by using a physical and chemical elicitor was provided.
[26] The object of the present invention is to provide a method of culturing adventitious root comprising the steps of:
[27] - steriliang and inoculating a plant to induce and to multiply callus;
[28] - inducing and multiplying the adventitious root from the callus; and
[29] - culturing the adventitious root in a bioreactor and harvesting it,
[30] characterized in that,
[31] (1) l/2mMS culture mediun [modified MS medium 1/2 MS (NH 4 +:NO3 =5:25) + sucrose 50g/L + IBA 2.0mg/L] is used as a culture mediun,
[32] (2) inoculum size of the adventitious root is 5~10g/L,
[33] (3) air supply amount is 0.05~0.3vvm,
[34] (4) culture period is from 3 to 5 weeks, and
[35] (5) culture temperature is between 15-250C.
[36] According to said culture method, it is preferred that the inoculun size of the adventitious root is 7g/L, air supply amount is 0.1 vvm, culture period is 5 weeks and culture temperature is 2O0C.
[37] The present invention further includes the culture method of the adventitious root which includes l/2mMS culture medium is further added 2 weeks after the start of the culture, in order to improve the growth of the adventitious root.
[38] The present invention further includes the culture method of the adventitious root, which includes that from the initial point of the growth the adventitious roots are treated everyday in a growth chamber having a controlled light intensity of 30 μmolm ~2 s"1 with an illumination cycle of 3h/21h (light/dark) and then cultured, in order to increase the content of the polyphenolic compound, which is a secondary metabolite of the adventitious root. [39] The present invention further includes the culture method of the adventitious root, which includes culturing under UV-A fluorescent lamp (2OW, Sankyo Denki, Japan) as a physical elicitor, placed 50cm above the bioreactor, everyday for Ih for one week from the fourth week of the culture, in order to increase the content of the polyphenolic compound which is a secondary metabolite of the adventitious root.
[40] The present invention further includes the culture method of the adventitious root, which includes treating the roots with nitrogen monoxide at 100 μmol as a chemical elicitor at the fourth week of the culture and then culturing them, in order to increase the content of the polyphenolic compound which is a secondary metabolite of the adventitious root.
[41] The present invention further includes the culture method of the adventitious root, which includes treating the roots with salicylic acid at 200 μmol as a chemical elicitor at the fourth week of the culture, in order to increase the content of the polyphenolic compound which is a secondary metabolite of the adventitious root.
[42] The present invention further includes the culture method of the adventitious root, characterized in that the plant is selected from ginseng, wild ginseng or cultivated wild ginseng.
[43] The present invention further includes the culture method of the adventitious root, characterized in that the plant is Echinacea purpurea.
[44] The present invention further includes the culture method of the adventitious root, characterized in that the adventitious root cultured at large scale is genetically stable as being identical with the parent in terms of the chromosome polyploidy and its number.
[45] The present invention further provides adventitious root that is cultured by the culture method of the present invention.
[46] More detailed explanation can be obtained through the following examples which are set forth herein to illustrate the present invention, but the scope of the present invention is not to be construed as being limited by the examples.
[47]
[48] Example 1. The induction of the callus and the adventitious root
[49] The Echinacea purpurea used in the present invention was seedling which had been grown from the germinated seed purchased from Canada. After cutting a stem and a leaf of the plant with an appropriate size for the surface sterilization before inoculation, they were immersed in 70% ethanol for 30 second, mixed with 4% sodium hypochlorite for 5 minutes, sterilized, washed with sterile water, and finally made in small pieces (lxθ.5xθ.3cm) which were then used as a culture material. [50] In order to induce the callus from a stem and a leaf of the plant body germinating from the seed, MS (Murashinge and Skoog, 1962) basal medium containing the naphthalene acetic acid (NAA) 2mg/L, the growth regulator of the benzyladenine (BA) 0. lmg/L, sucrose of 30g/L, and the gelite of 2.0g/L were inoculated and cultured for 8 weeks in a dark incubation room at 22+10C. To induce adventitious root from the obtained callus, sucrose of 30g/L, the gelite of 2.0g/L, and the NAA of 0.5mg/L as a growth regulator were added to the MS basal medium and then incubated in the dark incubation room at 22+10C for 8 weeks. Each of the culture medium was adjusted to pH 5.8 by using IN NaOH, sterilized for 20 minutes using autoclave at 1210C, and then aliquoted to a plastic cell culture dish 30m# each.
[51] After cutting the apical region of the induced adventitious root with about lcm, it was inoculated in the liquid culture medium, in which the IBA lmg/L and sucrose 50g/L were added in 1/2MS culture medium. The 14L culture medium was added to the airlift type balloon-like bubble bioreactor (balloon type bubble bioreactor, BTBB) of which the total volume is 2OL, and incubated under dark condition at 22+10C for 4 weeks.
[52]
[53] Example 2. Determination of factors affecting the growth of the adventitious root during culture in bioreactor
[54] The culture medium of 4L was added in the airlift type BTBB of the total volume 5L and incubation was performed for 5 weeks at 22+10C in dark condition. Each treatment was carried out in duplicate. The culture medium used in this experiment was the culture medium consisting of the mMS culture medium [the modified MS medium: 1/2 MS(NH4 +:NO3 =5:25) + sucrose 50 g/L + IBA 2.0mg/L). The effects of the mediun concentration, the inoculum size of the adventitious root, the air supply amount, and the culture period were investigated with respect to the growth of the adventitious root and the content of phenol, flavonoid, and caffeic acid derivatives, respectively.
[55] For each step, the method for measuring the adventitious root growth, the content of phenol, flavonoid and caffeic acid is as follows.
[56] After selecting the harvested adventitious roots by sifting them through the stainless mesh, the culture medium was separated off, moisture was removed with a clean tissue, and then the weight of the raw adventitious root was measured. After drying the root at 6O0C for 2 days, the dry weight was measured (the growth rate=[(the dry weight of the grown adventitious root) -(the dry weight of the adventitious root when it was initially inoculated)]/(the dry weight of the adventitious root when it was initially in- oculated)).
[57] The content of phenol, flavonoid, and caffeine acid derivative etc. was analyzed using an extract of the dried plant body obtained by using 80% methanol. The phenol content was measured by 760nm absorbance of Folin-Ciocalteu reagent against gallic acid standards. The flavonoid was measured with an absorbance at 510nm using catechin standards. The HPLC analysis condition for measuring the content of the caffeic acid derivatives includes the use of water and acetonitrile as a mobile phase and the XTerra
(R>
RP 18 column with the reading at 330nm absorbance.
[58] <2-l. Effect of the concentration of the culture medium on the growth of the adventitious root in bioreactor> [59] The adventitious root (7.Og) was inoculated to the 4 L culture media which comprises 1/4, 1/2, 3/4, and 1 MS concentration each in 5L bioreactor. The bioreactor was equipped with an air pressure compressor supplying air, an air condenser controlling the temperature of air, a filter and a pneumatic drier for removing impurity. The air dosage was controlled as 0.1 vvm after passing through the air flow control apparatus and fine filter. The culture was performed in duplicate treatment for 5 weeks at 22+10C under dark condition.
[60] Table 1 [Table 1] [Table ] Growth of adventitious root and content of polyphenolic compounds
Figure imgf000009_0001
[61] The analyzed values are verified for the significance by Duncan's multiple test using SAS(SAS Institute, Cary, NC) program.
[62] Table 2 [Table 2] [Table ] Caffeic acid derivatives content
Figure imgf000010_0001
[63] As a result of investigating the concentration of inorganic materials required for culture medium for the growth of the Echinacea purpurea adventitious root, it was found that the 1/2 MS treatment is the most effective as the fresh weight (e.g., weight of raw and fresh root) 73.6g/L, the dry weight 10.03g/L, the total phenol content 61.14mg/g DW, the total flavonoid content 38.30mg/g DW, and total caffeic acid derivative content 38.28mg/g DW. Therefore, the concentration of the inorganic materials required for the culture mediun for the growth of the Echinacea purpurea adventitious root was found to be 1/2MS (table 1 and table X).
[64] <2-2. Effect of adventitious root inoculum size> [65] The culture condition was the same as above Examples, and the adventitious root having respective concentration of 2.5, 5.0, 7.0, 10.0, 15.0g/L was inoculated in a bioreactor containing 4L of culture medium, followed by culturing.
[66] Table 3
[Table 3]
[Table ]
Growth of adventitious root and content of polyphenolic compounds
Figure imgf000011_0001
[67] The analyzed values are verified for the significance by Duncan's multiple test using SAS(SAS Institute, Cary, NC) program. [68] Table 4 [Table 4] [Table ] Caffeic acid derivatives content
Figure imgf000011_0002
[69] As a result of culture with different inoculum size in 5L bioreactor, a certain tendency was found that both of the fresh weight and dry weight increased as the inoculum size increased. But, at more than 7.0g/L of inoculum size, the content of all secondary metabolite including the total caffeine acid derivatives, etc. tended to decrease. From these results, the optimum inoculum size of the Echinacea adventitious root was found to be 7.0g/L, with the fresh weight 67.2g/L, the dry weight 10.80g/L, the total caffeic acid derivatives content 37.42mg/g DW (table 3 and table 4). [70] <2-3. Effect of air supply amount in bioreactor> [71] To determine the effect of air supply amount in bioreactor on the growth of the adventitious root, the air supply amount was varied at 0.05, 0.1, 0.2, 0.3, 0.05-0.1, 0.05-0.2 or 0.05-0.3 vvm, respectively. The bioreactor and culture condition were the same as the above Examples, and the inoculun size of adventitious root was 7.0g/L.
[72] Table 5 [Table 5] [Table ] Growth of adventitious root and content of polyphenolic compounds
Figure imgf000012_0001
[73] The analyzed values are verified for the significance by Duncan's multiple test using SAS(SAS Institute, Cary, NC) program. [74] Table 6
[Table 6]
[Table ]
Caffeic acid derivatives content
Figure imgf000013_0001
[75] Among the treatments with various air supply amount for the growth of the
Echinacea purpurea adventitious root, the air supply amount more than 0.1 vvm inhibited the growth of the adventitious root and content of a secondary metabolite. In this regard, as the growth of the adventitious root progressed, the fresh weight and dry weight, and the amount of secondary metabolites increased a little bit for the group to which the air supply was slowly increased up to 0.3 vvm. However, there was no significant difference compared to the group treated with the air supply amount fixed at 0.1 vvm. Therefore, the air supply amount suitable for the growth of the Echinacea purpurea adventitious root was found to be 0.1 vvm, with the fresh weight 70.1g/L, the dry weight 9.06g/L, the total caffeic acid derivatives content 37.39mg/g DW (table 5 and the table 6).
[76] <2-4. Change in the growth of the adventitious root according to the culture period in bioreactor>
[77] The inoculum size of adventitious root was 7.OgL"1 and the air supply amount was 0.1 vvm. The composition of supplied air was the same as atmospheric air (N 2 78%, O2 20.8%, CO2 0.03%, Ne, He) and the total culture period was 8 weeks. The content of phenolic compound and caffeic acid derivatives, pH of the medium, EC(electrical conductivity), and the content of sugar, cation and anion were analyzed with the interval of one week. The culture condition was the same as the above Example 2-1. [78] As a result of investigating the growth of the adventitious roots and a secondary metabolite content every week to follow the change of the growth of the adventitious root according to the bioreactor culture period and phenol content, it was found that, the fresh weight and dry weight drastically increased from week 1 to week 3 of culture, gradually increased from week 3 to week 5 and then the growth rate decreased starting from week 5 and nearly stopped at week 7 of the culture. While drastically increased from week 1 to week 3, a secondary metabolite content including the total phenol content, the total flavonoid content, the total caffeine acid derivative, etc. gradually increased from week 3 to week 5, and then the increment was reduced or their content was decreased from week 5. From this, the appropriate culture period of the Echinacea adventitious root is found to be five weeks in 5L bioreactor. The content of sucrose in the culture medium drastically dropped from the beginning of the culture to week 2, suggesting that it was degraded to glucose and fructose and then used by the plant. The content of cation and anion in the culture mediun drastically dropped from week 2 and then exhausted, causing an imbalance of the culture medium mesotrophy.
[79]
[80] Example 3. Effectsofadditionofthemediumhavingdifferentconcentration. different culturetemperatureandillumination cvcleonthegrowthof adventitious rootinbioreactor
[81] All experiments were performed using the culture medium of 4L in the airlift type
CTBB having total volume of 5L, and the culture was performed for 5 weeks at 22+10C under dark condition. Each treatment was carried out in duplicate. The culture medium used in the experiment consisted of l/2mMS culture mediun [modified MS medium: 1/2 MS(NH4 +:NCγ=5:25) + sucrose 50 g/L + IBA 2.0mg/L]. The effect of the addition of the mediun having different concentration, different culture temperature and illumination cycle on the growth of adventitious root in bioreactor on the growth of the adventitious roots, and the content of phenolic compounds, flavonoid, and caffeic acid derivatives were determined.
[82] <3-l. Effectof addition of the culture medium having different concentration on the growth of adventitious root>
[83] After the culture under the above culturing condition, the culture mediun with concentration of 1/4, 1/2, 3/4 or 1 MS was further added at week 2 and week 3 and their effect on the growth of the adventitious root was investigated.
[84] Table 7 [Table 7]
[Table ]
Growth of adventitious root and content of polyphenolic compounds
Figure imgf000015_0001
[85] The analyzed values are verified for the significance by Duncan's multiple test using
SAS(SAS Institute, Cary, NC) program. [86] Table 8 [Table 8]
[Table ]
Caffeic acid derivatives content
Figure imgf000016_0001
[87] The culture medium having different concentration was added in week 2 and week 3, in order to increase the growth of the adventitious root and content of a secondary metabolite. The fresh weight and dry weight were higher for the group to which the culture medium was further added compared to the control group. For the group to which a lower amount of the culture medium was added, the content of a secondary metabolite showed a level equivalent to the control group, while it is lower for the group to which a higher amount of the culture medium was added compared to the control group. The treatment group showing excellent fresh weight, dry weight, and total secondary metabolite content was 1/2MS culture mediun group wherein the 1/2 MS medium was added at week 2, with the fresh weight 83.1g/L (1.27 times), the dry weight 14.76g/L (1.5 times), the total phenol content 59.21mg/g DW, the total flavonoid 34.19mg/g DW, and the total caffeic acid derivative content 36.15mg/g DW which are the equivalent content with the control (table 7 and table 8).
[88] <3-2. Effect of culture temperature on the growth of adventitious root> [89] 2L of l/2mMS culture medium[modified MS medium: 1/2 MS(NH 4 +:NO3 =5:25) + sucrose 50g/L + IBA 2.0mg/L] 2L was added to the 3L of bioreactor and the adventitious root was cultured at different temperatures of 10, 15, 20, 25 and 3O0C.
[90] Table 9 [Table 9] [Table ] Growth of adventitious root and content of polyphenolic compounds
Figure imgf000017_0001
[91] The analyzed values are verified for the significance by Duncan's multiple test using SAS(SAS Institute, Cary, NC) program. [92] Table 10
[Table 10]
[Table ]
Caffeic acid derivatives content
Figure imgf000018_0001
[93] The growth of the Echinacea adventitious root was found to be sensitive to the temperature. At 2O0C, all of the fresh weight (65.5g/L), dry weight (10.37g/L), the total phenol content (61.5mg/g DW), total flavonoid content (38.0mg/L), and the total caffeic acid derivative content (38.28mg/g DW) were the highest. At 1O0C, the growth was the lowest. Moreover, in order to investigate the activity of the antioxidant enzyme according to temperature, the activity of SOD, APX, CAT, G-POD, and DPPH were measured after incubation for 5 days at different temperatures of 10, 25, 20, 25 and 3O0C.
[94] While the activity of the enzyme SOD was not much different among different temperature conditions, the activity of APX showed a tendency to decrease as the temperature increased. The activity of G-POD was lowest at 2O0C and 250C and the activity of CAT was the highest at 1O0C. The radical eliminating activity of DPPH was the highest at the optimum growth temperature (Table 9, Table 10, and Fig. 2).
[95] After crushing the adventitious root (1.Og, obtained after culturing for five weeks) by using liquid nitrogen with phosphoric acid buffer followed by centrifugation, the supernatant was taken for preparing a crude enzyme solution for the SOD, APX, CAT, the G-POD analysis.
[96] The activity of SOD was measured by reading an absorbance at 560nm after irradiating with a fluorescent lamp the crude enzyme solution in a transparent tube, and then the measured absorbance was compared to that of the control. The G-POD activity was measured by following the decrease of an absorbance at 470nm in accordance with the oxidation of a guaiacol under the presence of H2O2. The activity of the CAT (catalase) was measured at 240nm by following the concentration reduction of H2O2 when the enzyme extract of predetermined amount was added to the reaction solution comprising H 2O2. The activity of the APX (Ascorbate peroxidase) was measured by the absorbance decrease at 290nm in accordance with the oxidation of the ascorbic acid as H2O2 was added to the crude enzyme solution. The radical eliminating ability was measured by using DPPH method. Specifically, after DPPH being added to the methanol extract of the dry adventitious root and keeping the mixture for 10 minute, the absorbance decrease at 520nm was measured.
[97] <3-3. Effect of illumination cycle> [98] 2L of l/2mMS culture medium[modified MS medium: 1/2 MS(NH 4 +:NO3 =5:25) + sucrose 50g/L + IBA 2.0mg/L] was added to the bioreactor (3L) and the adventitious root was cultured under the condition including the temperature of 2O0C, illumination intensity of 30μmol nrV1- and illumination cycle of 0h/24h(light/dark), 3h/21h, 6h/18h or 12h/12h, respectively.
[99] Table 11 [Table 11] [Table ] Growth of adventitious root and content of polyphenolic compounds
Figure imgf000019_0001
[100] The analyzed values are verified for the significance by Duncan's multiple test using
SAS(SAS Institute, Cary, NC) program. [101] Table 12 [Table 12]
[Table ]
Caffeic acid derivatives content
Figure imgf000020_0001
[102] As a result of the culturing the adventitious roots with a different illumination cycle for 5 weeks, it was found that the fresh weight and the dry weight increased in the treatments of 3h/21h and 6h/18h cycle than the control of 0h/24h, but there was no significant difference. The content of a secondary metabolite was higher than the control and 3h/21h treatment group was found to be the best with the total phenol content 56.64mg/g DW (1.07 times), the total flavonoid 36.41mg/g DW (1.26 times), and the total caffeic acid derivative content 47.27mg/g DW (1.23 times). The illumination cycle of 12h/12h (i.e., longer exposure to light) showed a tendency that both the growth of the roots and a secondary metabolite content decreased compared to the control group (Table 11, Table 12).
[103] [104] Example 4. Effectofelicitoronthegrowthofadventitiousrootandthe content ofphenolic compounds andcaffeicacidderivatives
[105] To maximize the growth of the adventitious root in bioreactor and the accumulation of secondary metabolites, two- stage culture was performed. The adventitious root was cultured for 4 weeks in a bioreactor to achieve a constant level of growth, and then further cultured for one more week starting from week 5 during which an elicitor was applied.
[106] <4- 1. The effect of physical elicitor, UV- A>
[107] 2L l/2mMS [modified MS medium: 1/2 MS(NH 4 +:NO3 =5:25)+ sucrose 50g/L +
IBA 2.0mg/L] culture medium was added to 3L bioreactor and the adventitious root of Echinacea was cultured for five weeks at 2O0C under dark condition. The roots were irradiated every day for 0h,0.5h,lh,4h, respectively by using an UV-A fluorescent lamp (2OW, Sankyo Denki, Japan) during week 4, and the lamp was placed 50 an above the bioreactor. The experiment was carried out in duplicate.
[108] Table 13 [Table 13] [Table ] Growth of adventitious root and content of polyphenolic compounds
Figure imgf000021_0001
[109] The analyzed values are verified for the significance by Duncan's multiple test using
SAS(SAS Institute, Cary, NC) program. [110] Table 14
[Table 14]
[Table ]
Caffeic acid derivatives content
Figure imgf000021_0002
[111] For the UV-A treatment group, the fresh weight, dry weight, total phenol content, total flavonoid content, and total caffeic acid derivative content were all increased compared to the control group. Among them, Ih treatment group was the best, with the fresh weight of 67.7g/L, dry weight of 10.59g/L, total phenol content of 61.72rηg/g DW(1.20 times), total flavonoids content of 36.52mg/g DW(1.14 times), and total caffeic acid content of 42.38mg/g DW(1.24 times). (Table 13, Table 14).
[112] <4-2. Effect of nitrogen monoxide as a chemical elicitor> [113] 4L of l/2mMS culture medium[modified MS medium: 1/2 MS(NH 4 +:NO3 =5:25) + sucrose 50g/L + IBA 2.0mg/L] was added to the 5L of bioreactor and the adventitious root was cultured for five weeks at 2O0C under dark condition. Nitrogen monoxide(NO) was treated with the concentrations of 0, 50, 100, 250 or 500μmol at week 4 of the culture and each treatment was carried out in duplicate.
[114] Table 15 [Table 15] [Table ] Growth of adventitious root and content of polyphenolic compounds
Figure imgf000022_0001
[115] The analyzed values are verified for the significance by Duncan's multiple test using
SAS(SAS Institute, Cary, NC) program. [116] Table 16 [Table 16]
[Table ]
Caffeic acid derivatives content
Figure imgf000023_0001
[117] For all the groups treated with NO, fresh weight and dry weight were higher than the control while the growth was similar to the control. A secondary metabolite content was the highest in the lOOμmol treatment, with the fresh weight 69.8g/L, dry weight 11.31g/L, total phenolic compounds 61.07mg/g DW(1.06 times), total flavonoid content 39.93mg/g DW(1.07 times), total caffeic acid 43.55mg/g DW(1.23 times). The treatment with high concentration NO reduced the content of secondary metabolites. SOD antioxidant enzyme activity was almost the same for all treatment groups, while G-POD antioxidant enzyme activity was the lowest for 50μmol group. APX enzyme activity was the highest for lOOμmol treatment group (Table 15, Table 16, figure 3).
[118] <4-3. Effect of salicylic acid>
[119] 4L of l/2mMS culture medium[modified MS medium: 1/2 MS(NH 4 +:NO3 =5:25) + sucrose 50g/L + IBA 2.0mg/L] was added to the 5L of bioreactor and the adventitious root was cultured for five weeks at 2O0C under dark condition. Salicylic acid was treated with the different concentrations of 200, 400, 600 or 800μmol at week 4 of the culture and each treatment was carried out in duplicate.
[120] Table 17 [Table 17]
[Table ]
Growth of adventitious root and content of polyphenolic compounds
Figure imgf000024_0001
[121] The analyzed values are verified for the significance by Duncan's multiple test using
SAS(SAS Institute, Cary, NC) program. [122] Table 18
[Table 18]
[Table ]
Caffeic acid derivatives content
Figure imgf000024_0002
[123] High concentration of salicylic acid inhibited the growth of adventitious root and secondary metabolite content, while low concentration of salicylic acid showed almost the same growth as the control group. Content of secondary metabolites was higher than the control group.
[124] Among the different treatment groups, 200μmol treatment group was found to be the best, with the fresh weight 71.5g/L, dry weight 9.32g/L, total phenolic compounds 60.69mg/g DW(1.29 times), total flavonoid content 32.84mg/g DW(1.05 times), total caffeic acid 40.36mg/g DW(LI l times) (Table 17 and Table 18).
[125]
[126] Example 5. MassproductionoftheEchinaceapurpureaadventitiousrootfor obtaining the secondarymetabolites
[127] For the mass production of the Echinacea adventitious root, the characteristic of a secondary metabolite content and genetic stability of the adventitious root were examined first. A secondary metabolite content was compared with that of three kinds of cultured roots that had been cultured in natural environment condition for three years, while the genetic stability was verified by examining the chromosome number and polyploidy.
[128] < 5- 1. HPLC analysis of the caffeic acid>
[129] Three Echinacea species that were used in the present analysis had been purchased from Canada and they were three-year-old cultivated product. Echinacea purpurea adventitious root was obtained from the culture in 2OL bioreactor by using an optimal culture medium. As to the caffeic acid derivative content, HPLC analysis condition includes the use of water and acetonitrile as a mobile phase and the XTerra
®
RP 18 column with the absorbance reading at 330nm. [130] Table 19
[Table 19] [Table ] HPLC analysis
Figure imgf000026_0001
[131] For the roots of E angustifolia, echinacoside was the only one detected of which content was 6.70mg/g DW. For the root of E pallida, caftaric acid 0.34mg/g DW, echinacoside 5.96mg/g DW, cichoric acid 0.66mg/g DW, total 6.97mg/g DW were detected. For the roots of E.purpuread, caftaric acid 2.39mg/g DW, cichoric acid 6.18mg/g DW, total 8.57mg/g DW were detected. For the adventitious roots of E. pupura, caftaric acid 4.39mg/g DW, chlorogenic acid 5.25mg/g DW, cichoric acid 24.48mg/g DW, total 34.12mg/g DW were detected, in which the content of caftaric acid is 1.84 times and cichoric acid is 3.96 times higher as compared with those found in the naturally cultivated E.purpurea. Chlorogenic acid was detected only from E. pupura (Table 19, and fig. 6).
[132] <5-2. Determination of chromosome polyploidy of Echinacea>
[133] In order to confirm any variation in the chromosome polyploidy of the adventitious root that had been subcultured for a long period of time in a bioreactor, its polyploidy level of the leaf was compared with that of the same species grown in natural state. The adventitious root and leaf were cut into a size of lcm and of 0.5αnx0.5 cm (widthxlength), respectively, and then treated to extract nucleus. Ploidy analyzer (Partec, Germany) was used for the detection.
[134] As a result, the peaks were found at the same position for both of the adventitious root and the parent plant, suggesting that there was no change at nucleus level by the long-term culture of the adventitious root of Echinacea. Further, having the same chromosome polyploidy as the parent plant, the adventitious root of Echinacea was found to be genetically stable (fig. 4).
[135] <5-3. Chromosome nunber analysis of the adventitious root>
[136] In order to detect the chromosome, the tip of the roots were collected, pre-treated in a saturated aqueous solution of 1-bromonaphthalene for 22-24 hours at 40C, fixed by using a fixing solution (1:3, glacial acetic acid: ethanol, vV), softened in IN HCl at 6O0C for 6 minutes, and then dyes in Feulgen solution to prepare a specimen slide. Thus prepared slide was analyzed under an optical microscope. As a result, it was found that the chromosome number of the adventitious root which had been cultured according to the method of the present invention was 2X=22. This is in good agreement with the number suggested by Battaglia in his work of «CHROMOSOME ATLAS OF FLOWERING PLANTS», describing that the number of chromosomes in Echinacea purpurea is 22 (1947). Therefore, the genetic stability of the adventitious root of Echinacea as prepared according to the method of the present invention is confirmed again (fig. 5).
[137] <5-4.The adventitious root culture in high-capacity bioreactor>
[138] The mass production of the Echinacea adventitious root was carried out by using 300L, 600L l/2mMS [modified MS medium: 1/2 MS(NH 4 +:NO3 =5:25) + sucrose 50g/L + IBA 2.0mg/L] medium added to 500L, IOOOL balloon-type and drum type bioreactor, with 7g/L inoculum size at 220C, under dark condition for 50 days. Consequently, the growth of the adventitious root and caffeic acid content comprised therein were determined.
[139] Table 20 [Table 20] [Table ] Growth of adventitious root and content of polyphenolic compounds
Figure imgf000027_0001
[140] From 2OL bioreactor, the Echinacea adventitious root was harvested with the fresh weight 1.07Kg, the dry weight 0.14Kg, and the total caffeic acid derivative content 36.68mg /g DW. From 500L balloon type bioreactor, the Echinacea adventitious root was harvested with the fresh weight 26.80kg, the dry weight 3.62Kg, and the total caffeic acid derivative content 26.84mg/g DW. From IOOOL bioreactor, the adventitious root was grown to the fresh weight 41.02Kg, the dry weight 5.10kg, and the caffeic acid derivative content 32.03mg/g DW. These results indicate that the adventitious root of Echinacea can be successfully grown at large-scale in a bioreactor without any inhibition on growth and also various useful secondary metabolites can be produced with relatively high content in short period of time (Table 20).

Claims

Claims [1] A method of culturing adventitious root comprising the steps of: - sterilizng and inoculating a plant to induce and to multiply callus; - inducing and multiplying the adventitious root from the callus; and - culturing the adventitious root in a bioreactor and harvesting it, characterized in that,
(1) l/2mMS culture medium [modified MS medium: 1/2 MS (NH 4 +:NO3 =5:25) + sucrose 50g/L + IBA 2.0mg/L] is used as a culture medium,
(2) inoculum size of the adventitious root is 5~10g/L,
(3) air supply amount is 0.05~0.3vviη
(4) culture period is from 3 to 5 weeks, and
(5) culture temperature is between 15-250C.
[2] The culture method of the adventitious root according to claim 1, characterized in that inoculum size of the adventitious root is 7g/L, air supply amount is 0.1 vvm, culture period is 5 weeks and culture temperature is 2O0C.
[3] The culture method of the adventitious root according to claim 1, characterized in that it further comprises an addition of l/2mMS culture medium two weeks after the start of the culture, in order to improve the growth of the adventitious root.
[4] The culture method of the adventitious root according to claim 1, characterized in that from the initial point of the growth the adventitious roots are treated everyday in a growth chamber having a controlled light intensity of 30 μmol m ~2s 1 with an illumination cycle of 3h/21h (light/dark) and then cultured, in order to increase the content of the polyphenolic compound, which is a secondary metabolite of the adventitious root.
[5] The culture method of the adventitious root according to claim 1, characterized in that it further comprises the culture under UV-A fluorescent lamp (20W) as a physical elicitor, placed 50αn above the bioreactor, everyday for Ih for one week from the fourth week of the culture, in order to increase the content of the polyphenolic compound which is a secondary metabolite of the adventitious root.
[6] The culture method of the adventitious root according to claim 1, characterized in that it further comprises treating the roots with nitrogen monoxide at 100 μmol as a chemical elicitor at the fourth week of the culture and then culturing them, in order to increase the content of the polyphenolic compound which is a secondary metabolite of the adventitious root. [7] The culture method of the adventitious root according to claim 1, characterized in that it further comprises treating the roots with salicylic acid at 200 μmol as a chemical elicitor at the fourth week of the culture, in order to increase the content of the polyphenolic compound which is a secondary metabolite of the adventitious root. [8] The culture method of the adventitious root according to claim 1, characterized in that the plant is selected from the group consisting of ginseng, wild ginseng and cultivated wild ginseng. [9] The culture method of the adventitious root according to claim 1, characterized in that the plant is Echinacea purpurea. [10] The culture method of the adventitious root according to claim 1, characterized in that the adventitious root cultured at large scale is genetically stable as being identical with the parent plant in terms of the chromosome polyploidy and its nunber. [11] Adventitious root which is cultured by the culture method according to anyone of claims 1 to 10.
PCT/KR2007/007021 2007-02-14 2007-12-31 Method of bioreactor culture of echinacea purpurea adventitious roots WO2008100016A1 (en)

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CN108605831A (en) * 2016-12-16 2018-10-02 江苏省中国科学院植物研究所 A kind of method of Wikstroemia indica tissue-cultured seedling Rapid Rooting
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IT201900004119A1 (en) * 2019-03-21 2020-09-21 Demethra Biotech S R L Phytocomplex and extract of meristematic cell line selected from Echinacea purpurea
EP3808172A1 (en) 2019-10-14 2021-04-21 University of Science and Technology Method of obtaining sprouts by way of somatic embryogenesis from fragments of leaf blades with echinacea purpurea l. (moench)

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CN102613080A (en) * 2012-03-31 2012-08-01 常熟市佳盛农业科技发展有限公司 Method for rapidly propagating wild ginseng
CN102771397A (en) * 2012-08-17 2012-11-14 成都市三禾田生物技术有限公司 Method for establishing adventitious root cultivation system of Psammosilene tuniceoides W. C. Wu et C. Y. Wu and expanding cultivation method of Psammosilene tuniceoides W. C. Wu et C. Y. Wu
CN103120124A (en) * 2013-02-04 2013-05-29 南京工业大学 Method for continuously obtaining plant secondary metabolites by tissue culture and device thereof
CN103477985A (en) * 2013-09-09 2014-01-01 华南农业大学 Regeneration culture medium and culture method for improving regenerated adventitious buds of echinacea explants
CN103477985B (en) * 2013-09-09 2015-08-19 华南农业大学 A kind of regeneration culture medium and cultural method improving echinacea purpurea explant regeneration indefinite bud
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CN103609440A (en) * 2013-11-02 2014-03-05 吉林农业大学 Application method of elicitor in hairy root of American ginseng
WO2015149136A2 (en) 2014-04-01 2015-10-08 Green2Chem S.A. Continuous plug flow production method for plant roots outside soil by movement of a growing medium
CN105265312A (en) * 2014-07-17 2016-01-27 廉美兰 Method for improving effective substance in reactor-cultured oplopanax elatus nakai adventitious roots by using elicitors
CN105265312B (en) * 2014-07-17 2017-12-01 廉美兰 A kind of method that active principle in bioreactor culture elatus Nakai adventitious root is improved using elicitor
CN108605831A (en) * 2016-12-16 2018-10-02 江苏省中国科学院植物研究所 A kind of method of Wikstroemia indica tissue-cultured seedling Rapid Rooting
CN108605831B (en) * 2016-12-16 2021-08-27 江苏省中国科学院植物研究所 Method for rapidly rooting tissue culture seedlings of wikstroemia indica
EP3502229A1 (en) 2017-12-22 2019-06-26 Evologic Technologies GmbH Inoculation vessel and bioreactor for hairy root cultures
CN108308033A (en) * 2018-04-24 2018-07-24 镇江市德尔生物制品研究所有限公司 Atractylis lancea callus tissue culture base and apply its Atractylis lancea callus induction technique
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CN108739377A (en) * 2018-04-28 2018-11-06 刘汉石 A kind of ginseng adventitious root inducing culture and its application
IT201900004119A1 (en) * 2019-03-21 2020-09-21 Demethra Biotech S R L Phytocomplex and extract of meristematic cell line selected from Echinacea purpurea
WO2020188535A1 (en) * 2019-03-21 2020-09-24 Demethra Biotech S.R.L. Phytocomplex and extract of a meristematic cell line selected from echinacea purpurea
EP3808172A1 (en) 2019-10-14 2021-04-21 University of Science and Technology Method of obtaining sprouts by way of somatic embryogenesis from fragments of leaf blades with echinacea purpurea l. (moench)

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