Method for promoting growth of undifferentiated plant cell state or increasing content of secondary metabolites using micro-currents

Abstract

The present invention relates to: a micro-current device capable of promoting the growth of undifferentiated plant cells and controlling the content of physiologically active substances and secondary metabolites in cells; and a plant cell culture method using same.

Description

Method for promoting the growth of undifferentiated plant cells or increasing the content of secondary metabolites using microcurrents

[0001] The present invention relates to microcurrent stimulation for promoting growth of undifferentiated plant cells during in vitro culture and increasing the content of physiologically active substances and secondary metabolites within undifferentiated plant cells, and a culture method using the same.

[0002]

[0003] Plants have been used throughout human history for thousands of years in various fields such as pharmaceuticals, food, pigments, and cosmetics, and even today, the majority of the world's population relies on plant-based products. Almost all plants are capable of callus culture, as wounded tissue forms undifferentiated cells, or callus cells. Plant tissue culture is an important technology in both basic science and commercial applications; recently, biotechnology has enabled plants to be cultured in vitro through callus culture, cell suspension culture, and organ culture, and furthermore, provides opportunities to easily produce desired plant-derived recombinant proteins and bioactive substances through genetic engineering.

[0004] Plant cells are characterized by high plasticity and respond sensitively to abiotic and biotic stresses, affecting intracellular bioactive substances and secondary metabolites. Abiotic stresses include oxidative stress, temperature stress, and light stress, which are internally mediated using jasmonic acid, salicylic acid, and their derivatives as signal transducers. This series of external responses induces enzymatic synthesis of secondary metabolites produced through plant metabolic processes, thereby promoting the formation of numerous bioactive substances such as flavonoids, alkaloids, terpenoids, thionines, phenylpropanoids, and polypeptides. These secondary metabolites can provide beneficial effects to humans and animals as raw materials for products such as medical, cosmetic, and food products. However, the production of raw materials using plant cells has not yet been actively industrialized due to profitability issues.

[0005] Meanwhile, bioelectricity plays various roles, such as cell regeneration and activation and pain reduction; in modern times, various therapies utilizing microcurrents are being researched and utilized due to these characteristics. Plant cells also possess intracellular or intercellular mechanisms utilizing bioelectricity, specifically microcurrents, and are involved in maintaining intracellular homeostasis and cell regeneration. Exogenous electrical stimulation is an efficient and environmentally friendly technology that can regulate plant growth and development by modifying cellular metabolism through interactions with hydraulic, chemical, and hormonal signals.

[0006] Since the current flowing within plant cells is very minute, voltage must be supplied to measure it, and electrical conductivity can only be measured using highly sensitive sensors. External electrical influences generate bioelectrical potentials within the plant, and because the potential difference varies depending on the characteristics of the plant tissue, specific micro-stimulations tailored to the tissue characteristics are required. Callus cells are undifferentiated plant tissues that possess histological characteristics different from plant tissues (flowers, leaves, stems, roots, etc.) and respond more sensitively to external electrical stimuli.

[0007] Meanwhile, most studies on promoting plant cell growth using existing electrical stimulation have focused on plants that are already differentiated, i.e., complete plants. There is very little evidence regarding the intracellular or intercellular mechanisms using microcurrents in callus cells, which are undifferentiated plant cells, and nothing is known yet. However, the present invention has confirmed that when a certain range of microcurrents is used in undifferentiated plant cells, it is possible to control plant cell growth and secondary metabolites.

[0008] After researching a culture method that uses microcurrents to promote plant growth and increase the content of physiologically active substances and secondary metabolites within plant cells, the inventors found that the growth rate of undifferentiated plant cells cultured in a liquid in vitro for two weeks with microcurrents according to the present invention was promoted by a maximum of 133.33% in Pink Pepper hemp plants and a minimum of 33.28% in Lifter hemp plants. Specifically, it was confirmed that the content of THC, which belongs to the cannabinoids of Sativa-type blue hemp plants, increased by more than 45.23%, and the content of CBD in hybrid-type Lifter hemp also increased by more than 247.36%. Additionally, it was confirmed that the content of Saponarin, Apigenin, and Quercetin, which belong to the saponin family of Soapwort plants, also increased, and the content of Chlorogenic acid in Tobacco plants also increased.

[0009] Furthermore, the culture method using microcurrent according to the present invention is based on the characteristics of microcurrent that do not affect the cell viability of undifferentiated plant cells, and the growth rate of plant cells increased even under a wide range of microcurrent intensity conditions. In particular, the present invention was completed by confirming that the growth rate of plant cells increased significantly in dependence on microcurrent intensity and treatment time under specific conditions.

[0010]

[0011] The present invention aims to solve the difficulties encountered from an industrial perspective due to profitability issues when producing raw materials using undifferentiated plant cells (callus) as described above. Specifically, it seeks to resolve the profitability problem by promoting the growth of undifferentiated plant cells (callus) and controlling the content of physiologically active substances and secondary metabolites contained within the cells.

[0012]

[0013] Accordingly, the objective of the present invention is to provide a method for promoting plant cell growth or increasing the content of secondary metabolites using microcurrent, comprising the step of stimulating plant cell callus derived from plant seeds or tissues with microcurrent during culture.

[0014] Another objective of the present invention is to provide a method for producing a plant cell callus in which plant cell growth is promoted or the content of secondary metabolites is increased using microcurrent, comprising the step of stimulating the plant cell callus derived from a plant seed or tissue with microcurrent during culture.

[0015] Another objective of the present invention is to provide a plant cell callus produced by the above method that promotes plant cell growth or has an increased content of secondary metabolites.

[0016] Another objective of the present invention is to provide a method for culturing undifferentiated plant cells using microcurrents for promoting plant cell growth or increasing the content of secondary metabolites.

[0017]

[0018] The present invention promotes the growth of undifferentiated plant cells, namely calluses, using microcurrents and regulates physiologically active substances and secondary metabolites within the cells, thereby increasing profitability in terms of raw material production using plant cells. By improving profitability, high industrial value can be obtained in a wide range of industrial fields, such as pharmaceuticals, food, and cosmetics, through the production of plant cell-derived raw materials.

[0019]

[0020] FIG. 1 is a schematic diagram of a microcurrent stimulation according to one embodiment of the present invention.

[0021] FIG. 2 is a figure illustrating a method for inducing callus in plant seeds and tissues according to one embodiment of the present invention.

[0022] FIGS. 3a and 3b are figures showing changes in plant cell growth rate and changes in concentration and content of physiologically active substances within plant cells according to current intensity when microcurrent stimulation is applied to a Sativa-type hemp callus according to one embodiment of the present invention.

[0023] FIGS. 4a and 4b are figures showing changes in plant cell growth rate and changes in concentration and content of physiologically active substances within plant cells according to current intensity when microcurrent stimulation is applied to a hybrid type Lifter hemp callus according to one embodiment of the present invention.

[0024] FIGS. 5a and 5b are figures showing changes in plant cell growth rate and changes in concentration and content of physiologically active substances within plant cells according to current intensity when microcurrent stimulation is applied to a hybrid type Pink Pepper hemp callus according to one embodiment of the present invention.

[0025] FIGS. 6a and 6b are figures showing the changes in plant cell growth rate, concentration and content of physiologically active substances within plant cells according to the current intensity when microcurrent stimulation is applied to soapwort callus according to one embodiment of the present invention.

[0026] FIG. 6c is a figure showing the results of high-performance liquid chromatography (HLPC) analysis showing the change in the content of physiologically active substances in plant cells according to the treatment time when microcurrent stimulation is applied to soapwort callus according to one embodiment of the present invention.

[0027] FIG. 7 is a figure showing the change in growth rate and active substance content according to the current intensity when microcurrent stimulation is applied to tobacco callus according to one embodiment of the present invention.

[0028] FIG. 8 is a figure showing changes in the expression of cell growth-related genes (CsRBR) and cannabinoid biosynthesis-related genes (CsCBDAS, CsTHCAS) according to the current intensity when microcurrent stimulation is applied to Cheongsam and Lifter hemp calluses according to one embodiment of the present invention.

[0029]

[0030] The present invention will be described in more detail below.

[0031]

[0032] Meanwhile, each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions provided below.

[0033] Furthermore, a person skilled in the art can recognize or identify a number of equivalents to the specific embodiments of the present invention described in this invention using only ordinary experiments. In addition, such equivalents are intended to be included in the present invention.

[0034] Furthermore, numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated by reference into this specification in their entirety to more clearly explain the state of the art to which the present invention pertains and the content of the present invention.

[0035]

[0036] To achieve the above objectives, one aspect of the present invention provides a method for promoting plant cell growth or increasing secondary metabolite content using microcurrent, comprising the step of stimulating plant cell callus derived from plant seeds or tissues with microcurrent during culture.

[0037] In the present invention, the microcurrent may refer to electrical stimulation utilized in various fields, including beauty and medicine, along with high-frequency and low-frequency.

[0038] The term "micro-current" in this specification refers to a very minute current in the microampere (µA) range that is similar to the bioelectric current flowing within the body and does not stimulate sensory nerves, so the flow of the current is barely felt. It is known that when the above micro-current is applied to damaged cells, it can promote the production of ATP in the cells and aid in the recovery of damaged tissues, and can be used for various purposes such as skin regeneration, pain relief, or wound healing. Furthermore, the term "high-frequency" refers to a high-frequency current that can generate deep heat within the body when stimulating the human body, and can allow the generated heat to reach deep into the skin. It is known that the heat generated within the body by stimulation by the above high-frequency current can revitalize blood circulation and metabolism, and stimulate collagen-producing cells in the dermis layer to improve skin elasticity or reduce wrinkles. In addition, the term "low-frequency" can refer to a current with a low frequency in the unit of mA (milliampere) that, when stimulated by the human body, directly stimulates muscles and nerves to cause repeated contraction and relaxation of the muscles. It is known that stimulation by the low-frequency can deliver direct electrical signals to the muscles to artificially move them, thereby relaxing the muscles, or help with blood circulation and stimulate nerves to alleviate pain. Furthermore, there is a difference in that microcurrent is a direct current (DC) in which the magnitude and direction of the current do not change over time and remain constant, whereas high frequency or low frequency is an alternating current (AC) in which the magnitude and direction of the current change over time.Therefore, since the above-mentioned microcurrent differs from high frequency or low frequency in terms of frequency and current, and thus the operating principle and effect during biostimulation also differ, the microcurrent used in the present invention must be clearly distinguished from high frequency or low frequency.

[0039] In the present invention, the microcurrent may be applied by stimulating the plant cell callus with an electric current of 5 to 60 μA intensity once a day in a liquid medium using an electrophoresis device, but is not limited thereto.

[0040] According to one embodiment of the present invention, the microcurrent may be applied as a current of 0.2 to 100 mA or a voltage of 5 to 15 V and processed into a current of 5 to 1,500 μA.

[0041] Specifically, the current applied to the microcurrent is 0.2 to 100 mA, 0.2 to 70 mA, 0.2 to 60 mA, 0.2 to 50 mA, 0.2 to 40 mA, 0.2 to 30 mA, 0.2 to 25 mA, 0.2 to 20 mA, 0.2 to 15 mA, 0.2 to 10 mA, 0.2 to 5 mA, 0.5 to 100 mA, 0.5 to 70 mA, 0.5 to 60 mA, 0.5 to 50 mA, 0.5 to 40 mA, 0.5 to 30 mA, and 0.5 to 25 The current may be applied at a current of 10 to 15 μA, but is not limited to 1 mA, 0.5 to 20 mA, 0.5 to 15 mA, 0.5 to 5 mA, 1 to 100 mA, 1 to 70 mA, 1 to 60 mA, 1 to 50 mA, 1 to 40 mA, 1 to 30 mA, 1 to 25 mA, 1 to 20 mA, 1 to 15 mA, 1 to 10 mA, more specifically, 1 to 5 mA, and even more specifically, applied at a current of 1 to 3 mA to be processed as a current of 10 to 15 μA.

[0042] In addition, the microcurrent may be applied once a day at intervals of 5 to 60 minutes, and this may be repeated for 14 to 20 days.

[0043] Specifically, the microcurrent may be processed at intervals of 5 to 60 minutes, 5 to 50 minutes, 5 to 40 minutes, 5 to 30 minutes, 10 to 60 minutes, 10 to 50 minutes, 10 to 40 minutes, 10 to 30 minutes, 15 to 60 minutes, 15 to 50 minutes, 15 to 40 minutes, 15 to 30 minutes, 20 to 60 minutes, 20 to 50 minutes, 20 to 40 minutes, more specifically, 20 to 30 minutes, and even more specifically, 30 minutes, but is not limited thereto.

[0044] Specifically, the microcurrent may be processed repeatedly for 14 to 20 days, 14 to 18 days, 14 to 16 days, 15 to 20 days, 15 to 18 days, more specifically, 15 to 16 days, and even more specifically, 15 days, but is not limited thereto.

[0045] In the present invention, the plant may be a combination of the genus Cannabis (pp.), Saponarias (pp.), or Nicotianas (pp.), or the same.

[0046] In the present invention, the secondary metabolite may be a cannabinoid, a saponin, a chlorogenic acid, or a combination thereof, but is not limited thereto.

[0047] In the present invention, the cannabinoid may be tetrahydrocannabinol (THC), cannabidiol (CBD), cannabidiolic acid (CBDA), or a combination thereof, but is not limited thereto.

[0048] In the present invention, the saponin may be saponarin, apigenin, quercetin, or a combination thereof, but is not limited thereto.

[0049] In the present invention, the step of stimulating with microcurrent may be applied regardless of the type of bioreactor.

[0050]

[0051] Another aspect of the present invention provides a method for producing a plant cell callus in which plant cell growth is promoted or the content of secondary metabolites is increased by using microcurrent, comprising the step of stimulating the plant cell callus derived from a plant seed or tissue with microcurrent during culture.

[0052]

[0053] Another aspect of the present invention provides a plant cell callus produced by the above manufacturing method, in which plant cell growth is promoted or the content of secondary metabolites is increased.

[0054]

[0055] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings so that those skilled in the art can easily implement the present invention. Unless otherwise defined, terms used in this specification should be interpreted as generally understood by those skilled in the art.

[0056] The drawings and embodiments of this specification are intended to enable a person skilled in the art to easily understand and practice the invention. Content that may obscure the essence of the invention may be omitted from the drawings and embodiments, and the invention is not limited to the drawings and embodiments.

[0057] Throughout this specification, "%" used to indicate the concentration of a particular substance is (weight / weight)% for solid / solid, (weight / volume)% for solid / liquid, and (volume / volume)% for liquid / liquid, unless otherwise noted.

[0058] Unless otherwise specified, all numbers, values, and / or expressions used herein to denote ingredients, reaction conditions, and the content of ingredients shall be understood to be modified by the term “approximately” in all cases, as these numbers are essentially approximations reflecting the various uncertainties of measurement that occur in obtaining these values ​​among others.

[0059] And, where numerical ranges are disclosed in this specification, such ranges are continuous and, unless otherwise specified, include all values ​​from the minimum value of such range to the maximum value including the maximum value.

[0060] Furthermore, the term "or" in this specification is intended to mean an implied "or" rather than an exclusive "or." That is, where the combination or use of the configurations is not otherwise specified or is not evident from the context—i.e., where X includes A; X includes B; or X includes both A and B—"X includes A or B" may be applied to either of these cases.

[0061] Throughout this specification, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0062]

[0063] The present invention relates to an electrical stimulation method using microcurrent to promote the growth of plant cell calluses and to regulate physiologically active substances and secondary metabolites within plant cells.

[0064] The term "plant cell callus" in this specification refers to an undifferentiated, irregular mass of cells that can be obtained by inducing callus formation in plant tissue culture by adding the hormones auxin and cytokinin to the culture medium in appropriate proportions. The callus is divided into a relatively hard part and a soft part, and since the soft callus is easily dispersed in liquid, suspension culture is easy.

[0065] In the present invention, the experiment is easy because the electrical stimulation using microcurrent utilizes the principle of generating current in a liquid medium containing various ions that are electrolytes by applying voltage to an electrode immersed in a liquid medium.

[0066] In the present invention, cannabis is known to contain approximately 150 or more cannabinoid substances. Representative and relatively well-known CBD (Cannabidiol) is a substance having the structure of Chemical Formula 1 below and is used as a raw material for treating pediatric epilepsy. In some countries, it is utilized not only for medical purposes but also in various industrial fields such as food, cosmetics, and pet-related products. In addition to CBD, there are other cannabinoids with high potential industrial value, and numerous clinical studies are being conducted worldwide.

[0067]

[0068] In addition, THC (Tetrahydrocannabinol) is a substance having the structure of Chemical Formula 2 below and is the most representative psychoactive cannabinoid in cannabis, and is known to act on CB1 / CB2 receptors to produce various central and peripheral effects. Furthermore, CBN (Cannabinol) is a substance having the structure of Chemical Formula 3 below, and numerous studies are being conducted on it for improving sleep quality, stimulating appetite, and relieving pain. CBC (Cannabichromene) is a substance having the structure of Chemical Formula 4 below, and is highly valued for its potential to treat various diseases such as relieving depression and anxiety, controlling pain and inflammation, anticancer effects, and Alzheimer's and Parkinson's diseases, and many studies are being conducted on it. CBDA (Cannabidiolic acid) is a substance having the structure of Chemical Formula 5 below, is a precursor of CBN (Cannabinol), and is known to have anti-inflammatory, anti-depressant and anti-anxiety, and neuroprotective effects.

[0069]

[0070]

[0071]

[0072]

[0073] In the present invention, Saponarin contained in Soapwort is a substance having the structure of Chemical Formula 6 below, and is a natural saponin as a type of flavonoid glucoside. There are research results showing that it regulates gluconeogenesis and glucose absorption by activating AMPK in a calcium-dependent manner, and it is also known to have excellent anti-inflammatory efficacy by regulating the TLR4 / MD2 receptor in immune cells.

[0074]

[0075] In the present invention, chlorogenic acid contained in tobacco is a substance having the structure of Chemical Formula 7 below, and there are various research reports regarding its antioxidant activity, anti-inflammatory activity, blood sugar control, etc.

[0076]

[0077]

[0078] Example 1: Callus Suspension Culture and Microcurrent Stimulation

[0079] The inventors suspended the plant cell callus in a liquid medium and varied the treatment time and current intensity of the microcurrent stimulation to compare the growth of the callus and the content of physiologically active substances and secondary metabolites within the cell.

[0080] Callus was induced for hemp leaf explants in MS medium containing 4 mg / L TDZ (Thidiazuron), 2 mg / L NAA (1-Naphthaleneactic acid), 30 g / L Sucrose, and 8 g / L Plant agar for 4-6 weeks under a 16-hour photoperiod and 25°C conditions, and for soapwort and tobacco leaf explants in MS medium containing 1 mg / L 2,4-D, 30 g / L Sucrose, and 8 g / L Plant agar for 4-6 weeks under a 16-hour photoperiod and 25°C conditions.

[0081] For suspension culture, 2g of hemp callus was cultured in MS medium containing 2mg / L of TDZ (Thidiazuron), 1mg / L of NAA (1-Naphthaleneactic acid), and 15g / L of sucrose for 2 weeks in a dark room at 28℃, and 2g of soapwort and tobacco callus were cultured in MS medium containing 0.5mg / L of 2,4-D and 15g / L of sucrose for 2 weeks in a dark room at 28℃.

[0082]

[0083] First, 2g of the above callus was inoculated into a liquid medium, and for all suspension cultures, the pH was in the range of 5 to 6, and cultures were performed in a dark room and a light room. Microcurrent stimulation was applied once a day for 15 minutes for 2 weeks starting from the start of culture, and experiments were conducted by fixing the treatment time and varying the current intensity, or fixing the current intensity and varying the treatment time.

[0084]

[0085] Example 2: Comparison of callus growth induced by microcurrent stimulation

[0086] The suspension culture of Example 1 was carried out for 2 weeks, and the volume was measured in a flask. The sample was recovered, its weight before drying was measured using a microbalance, and it was vacuum freeze-dried for 24 to 48 hours. Afterward, the weight of the dried callus was measured using a microbalance, and this is shown in FIGS. 3a to 7a.

[0087] First, as can be seen in Figure 3a, in the case of the sativa-type Cheongsam hemp plant (C. sativa 'Cheungsam'), it was confirmed that the growth of plant cell calluses was promoted by microcurrent stimulation treatment compared to the control group, and that growth was significantly promoted by about 54% at 3 mA, and it was confirmed that the growth of hemp plant cell calluses can be effectively promoted in the range of microcurrent intensity greater than 0.5 mA and less than or equal to 40 mA.

[0088] As can be seen in Figure 4a, in the case of a hybrid type of cannabis plant (C. sativa 'Lifter'), it was confirmed that the growth of plant cell callus was promoted by microcurrent stimulation treatment compared to the control group, and that growth was significantly promoted by about 33% at 3 mA, and it was confirmed that the growth of cannabis plant cell callus can be effectively promoted in the range of microcurrent intensity greater than 0.5 mA and less than or equal to 40 mA.

[0089] As can be seen in Figure 5a, in the case of another type of hemp plant (C. sativa 'Pink Pepper'), it was confirmed that the growth of plant cell calluses was promoted by microcurrent stimulation treatment compared to the control group, and that growth was significantly promoted at 1 mA (about 133%) or 20 mA, and it was confirmed that the growth of hemp plant cell calluses can be effectively promoted in the range of microcurrent intensity from 0.2 mA to 100 mA.

[0090] As can be seen in Figure 6a, in the case of Soapwort (S. officinalis), the growth of plant cell callus was promoted by microcurrent stimulation treatment compared to the control group, and it was confirmed that growth was significantly promoted by about 68% at 3 mA, and it was confirmed that the growth of Soapwort plant cell callus can be effectively promoted in the range of microcurrent intensity from 0.2 mA to 70 mA.

[0091] As can be seen in Figure 7a, in the case of tobacco plants (N. tabacum), the growth of plant cell calluses was promoted by microcurrent stimulation treatment compared to the control group, and it was confirmed that growth was significantly promoted by about 50% at 0.5 mA, and it was confirmed that the growth of tobacco plant cell calluses can be effectively promoted in the range of microcurrent intensity from 0.2 mA to 70 mA.

[0092] In addition, regarding the growth rate of undifferentiated plant cells, it was confirmed that growth was promoted in the 20 mA to 40 mA range and then rapidly decreased. This range is predicted to be the pre-death burst range, and since it corresponds to a range where energy metabolism is overactivated by external stimuli such as stress, causing temporary impairment in growth and metabolic activity and leading to cell death, it was confirmed that microcurrent stimulation in the above range (20 mA to 40 mA) is not a stimulus for long-term growth promotion.

[0093]

[0094] Therefore, it was specifically confirmed that when microcurrent stimulation within a certain intensity range is applied, the growth of various plant cell calluses, including hemp, soapwort, and hemp plants, can be effectively promoted.

[0095]

[0096] Example 3: Comparison of concentration and total content of bioactive substances in plant cells induced by microcurrent stimulation

[0097] The samples for which dry weight measurement was completed in Example 2 above were extracted for 24 hours using 100% ethanol and methanol as solvents. Subsequently, the supernatant was filtered to remove impurities using a PVDF filter (0.2 μm) to obtain the samples. All samples were analyzed using HPLC (Agilent 1260 Infinity), and an Agilent Eclipse XDB-C18 chromatographic column (150 x 4.6 mm, 5 μm) was used. The concentration gradients of the mobile phase solvents were as follows for each plant.

[0098] Cannabis callus extract: water (containing 0.1% FA (Formic acid)) / acetonitrile was analyzed by varying the ratio to 30:70 during the initial 0 to 6 minutes, 23:77 during 17 to 28 minutes, and 30:70 during 28 to 30 minutes, with a flow rate of 1 ml / min and an atmospheric pressure of 400 bar. A UV detector (200 nm) was used as the detector.

[0099] Soapwort callus extract: Water (containing 0.1% FA (Formic acid)) / acetonitrile was mixed at a ratio of 95:5 for the initial 0 to 5 minutes, 0:100 for 5 to 20 minutes, and 95:5 for 20 to 23 minutes, maintained up to 25 minutes. The flow rate was 1 ml / min and the pressure was 400 bar for analysis. A UV detector (258, 290, 350 nm) was used.

[0100] Tobacco callus extract: water (containing 0.1% FA (Formic acid)) / acetonitrile was maintained at an 80:20 ratio for the initial 0 to 20 minutes, and analyzed at a flow rate of 1.2 ml / min and an atmospheric pressure of 400 bar. A UV detector (325 nm) was used as the detector, as shown in Figures 3b to 7b.

[0101] Specifically, in FIGS. 3b to 7b, the black bars represent the content of bioactive substances (THC, CBDA, saponarin, or CGA) contained in the same amount of callus, and the white bars represent the value calculated by considering both the content of bioactive substances and the plant cell growth rate (amount of callus in the corresponding group * content of the component). In this case, even if the plant cell growth rate is high, if the content of bioactive substances contained therein is low, it can be determined that the profitability is also low. Therefore, for high profitability, it is important to determine the intensity of the microcurrent or the treatment time that can satisfy an appropriate balance between the plant cell growth rate and the content of bioactive substances contained in the plant cells, that is, the size of the white bars can increase.

[0102] First, as can be seen in Figure 3b, in the case of tetrahydroxycannabinol (THC), one of the cannabinoids that are bioactive substances of the Sativa-type Cheongsam plant (C. sativa 'Cheungsam'), the total content (white bar) increased with microcurrent stimulation treatment compared to the control group, and it was confirmed that the concentration (black bar) and total content (white bar) within the plant cells increased significantly by about 45% when the applied current intensity was 5 mA, and it was confirmed that the total content (white bar) of the bioactive substance within the hemp plant cells can be effectively increased in the range of 0.2 mA to 5 mA.

[0103] As can be seen in Figure 4b, in the case of cannabidiolic acid (CBDA), one of the cannabinoids that are bioactive substances of the hybrid type of cannabis plant (C. sativa 'Lifter'), the concentration (black bar) and total content (white bar) within the plant cells increased with microcurrent stimulation treatment compared to the control group. It was confirmed that when the applied current intensity was 3 mA, the concentration (black bar) and total content (white bar) within the plant cells increased significantly by about 247%, and it was confirmed that the concentration (black bar) and total content (white bar) of the bioactive substance within the cannabis plant cells could be effectively increased in the range of 0.2 mA to 20 mA.

[0104] As can be seen in Figure 5b, in the case of tetrahydroxycannabinol (THC), one of the bioactive cannabinoids of another type of cannabis plant (C. sativa 'Pink Pepper'), the concentration (black bar) and total content (white bar) within the plant cells increased with microcurrent stimulation treatment compared to the control group. It was confirmed that the concentration (black bar) and total content (white bar) within the plant cells increased significantly by approximately 1,497% when the applied current intensity was 1 mA, and it was confirmed that the concentration (black bar) and total content (white bar) of the bioactive substance within the cannabis plant cells could be effectively increased in the range of 0.2 mA to 100 mA.

[0105] As can be seen in Figure 6b, in the case of saponarin, one of the saponins that are bioactive substances of the Soapwort plant (S. officinalis), the concentration (black bar) and total content (white bar) within the plant cells increased with microcurrent stimulation treatment compared to the control group, and it was confirmed that the concentration (black bar) and total content (white bar) within the plant cells increased significantly by about 85% when the applied current intensity was 1 mA. It was also confirmed that the concentration (black bar) and total content (white bar) of the bioactive substance within the Soapwort plant cells could be effectively increased in the range of 0.2 mA to 1 mA and 30 mA to 70 mA.

[0106] As can be seen in Figure 6c, in the case of quercetin, a bioactive substance of the soapwort plant (S. officinalis), the content in plant cells increased by approximately 81% when treated for 15 minutes with an applied current intensity of 1 mA compared to the control group, and in the case of apigenin, the content in plant cells increased by approximately 24% when treated for 30 minutes with an applied current intensity of 1 mA compared to the control group.

[0107] As can be seen in Figure 7b, in the case of chlorogenic acid (CGA), a bioactive substance of the tobacco plant (N. tabacum), the concentration (black bar) and total content (white bar) within the plant cells increased with microcurrent stimulation treatment compared to the control group. It was confirmed that the concentration (black bar) and total content (white bar) within the plant cells increased significantly by about 94% when the applied current intensity was 1 mA, and it was confirmed that the concentration (black bar) and total content (white bar) of the bioactive substance within the tobacco plant cells could be effectively increased in the range of 0.2 mA to 40 mA.

[0108]

[0109] Therefore, it was specifically confirmed that when microcurrent stimulation within a certain intensity range is applied, the concentration and total content of bioactive substances within various plant cells, including Cannabis, Soapwort, and Cannabis species, can be effectively increased.

[0110]

[0111] Example 4: Comparison of gene expression in plant cell metabolism induced by microcurrent stimulation

[0112] In Example 3 above, the relative gene expression of undifferentiated plant cells and cannabis plant (C. sativa 'Cheungsam' and 'Lifter') calluses after microcurrent stimulation was completed was compared.

[0113] Specifically, total RNA from cannabis plant (C. sativa 'Cheungsam' and 'Lifter') calluses was extracted after 2 weeks of suspension culture and microcurrent stimulation. RNA extraction was performed using Trizol reagent by the following method. Approximately 100 mg of callus tissue was placed in 1 mL of Trizol reagent and homogenized using a FastPrep-24 homogenizer (MP Biomedicals, USA) in a microtube containing beads. Subsequently, 200 µL of chloroform was added, and the mixture was centrifuged at 15,000 × g for 15 minutes. The supernatant was collected, the RNA was precipitated with isopropanol, washed with 75% ethanol, air-dried, and finally dissolved in 50 µL of RNase-free water. For cDNA synthesis, total RNA was reverse transcribed using Oligo dT 20 primers and AccuPower RT PreMix. In gene screening analysis, gene expression related to stress response regulation and secondary metabolite biosynthesis was investigated. The expression of CsRBR (Retinoblastomata-related protein), known as a negative regulator of cell division, was analyzed.

[0114] In addition, the expression of CsCBDAS (cannabidiolic acid synthase), which encodes the CBDA biosynthetic enzyme, and CsTHCAS (tetrahydrocannabinolic acid synthase), which encodes the THCA biosynthetic enzyme, was also investigated, and the CsEF1α (elongation factor 1 alpha) gene was used as an intrinsic control.

[0115] qRT-PCR was performed using PowerTrack SYBR Green MMSTER Mix, and primers were synthesized in mAcrogen. Primers were newly designed using NCBI Primer-BLAST, and the results are shown in Figures 8a to 8c.

[0116]

[0117] As can be seen in Figure 8a, in the case of the CsRBR gene in the cannabis plant (C. sativa 'Cheungsam'), it was confirmed that the expression level (black bar) increased when the applied current intensity was 0.5 mA to 1 mA compared to the control group, and in the case of the cannabis plant (C. sativa 'Lifter'), it was confirmed that the expression level (white bar) increased when the applied current intensity was 0.2 mA to 0.5 mA compared to the control group.

[0118] And, as can be seen in Figure 8b, in the case of the CsCBDAS gene in the cannabis plant (C. sativa 'Cheungsam'), it was confirmed that the expression level (black bar) increased when the applied current intensity was 0.2 mA to 100 mA compared to the control group, and in the cannabis plant (C. sativa 'Lifter'), it was confirmed that the expression level (white bar) increased when the applied current intensity was 0.5 mA compared to the control group.

[0119] In addition, as can be seen in Figure 8c, it was confirmed that the expression level (black bar) of the CsTHCAS gene in the cannabis plant (C. sativa 'Cheungsam') increased when the applied current intensity was 5 mA compared to the control group, and in the cannabis plant (C. sativa 'Lifter') it was confirmed that the expression level (white bar) increased when the applied current intensity was 0.5 mA compared to the control group.

[0120]

[0121] Therefore, it was specifically confirmed that when microcurrent stimulation within a certain intensity range is applied, the expression level of genes within plant cell metabolism in cannabis plants can be effectively increased.

[0122]

[0123] From the foregoing description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without altering its technical concept or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as including all modifications or variations derived from the meaning and scope of the claims set forth below and their equivalents, rather than from the detailed description above.

Claims

1. A method for promoting plant growth or increasing secondary metabolite content using microcurrent, comprising the step of stimulating plant cell callus derived from plant seeds or tissues with microcurrent during culture.

2. A method for promoting plant growth or increasing secondary metabolite content using microcurrent, wherein the microcurrent is applied to the callus once a day with an electric current of 5 to 60 μA intensity in a liquid medium using an electrophoretic device.

3. A method for promoting plant growth or increasing secondary metabolite content using microcurrents, wherein the plant is a Cannabis genus (Cannabisspp.), Saponarias genus (Saponariaspp.), Nicotianas genus (Nicotianaspp.), or a combination thereof, in accordance with claim 1.

4. A method for promoting plant growth or increasing the content of secondary metabolites using microcurrents, wherein the secondary metabolite is a cannabinoid, saponin, chlorogenic acid, or a combination thereof, in accordance with claim 1.

5. A method for promoting plant growth or increasing the content of secondary metabolites using microcurrents, wherein the cannabinoid in claim 4 is tetrahydrocannabinol (THC), cannabidiol (CBD), cannabidiolic acid (CBDA), or a combination thereof.

6. A method for promoting plant growth or increasing the content of secondary metabolites using microcurrents, wherein, in paragraph 4, the saponin is saponarin, apigenin, quercetin, or a combination thereof.

7. A method for producing a plant cell callus with increased plant growth or increased secondary metabolite content using microcurrent, comprising the step of stimulating the plant cell callus derived from a plant seed or tissue with microcurrent during culture.

8. A method for producing a plant cell callus in which plant growth is promoted or the content of secondary metabolites is increased using a microcurrent, wherein, in claim 7, the microcurrent is applied to the callus once a day with an electric current of 5 to 60 μA intensity in a liquid medium using an electrophoresis device.

9. A method for producing plant cell callus in which plant growth is promoted or the content of secondary metabolites is increased using microcurrent, wherein, in claim 7, the plant is a plant of the genus Cannabis spp., a plant of the genus Saponaria spp., a plant of the genus Nicotiana spp., or a combination thereof.

10. A method for producing plant cell callus in which plant growth is promoted or the content of secondary metabolites is increased using microcurrent, wherein, in claim 7, the secondary metabolite is a cannabinoid, saponin, chlorogenic acid, or a combination thereof.

11. A method for producing plant cell callus with increased plant growth or increased secondary metabolite content using microcurrent, wherein, in claim 10, the cannabinoid is tetrahydrocannabinol (THC), cannabidiol (CBD), cannabidiolic acid (CBDA), or a combination thereof.

12. A method for producing plant cell callus with increased plant growth or secondary metabolite content using microcurrent, wherein, in claim 10, the saponin is saponarin, apigenin, quercetin, or a combination thereof.

13. A plant cell callus produced by the method of any one of claims 7 to 12, in which plant growth is promoted or the content of secondary metabolites is increased.

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