CN116326478A - Glycyrrhiza glabra seedling culture medium and application thereof - Google Patents

Glycyrrhiza glabra seedling culture medium and application thereof Download PDF

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CN116326478A
CN116326478A CN202310244299.4A CN202310244299A CN116326478A CN 116326478 A CN116326478 A CN 116326478A CN 202310244299 A CN202310244299 A CN 202310244299A CN 116326478 A CN116326478 A CN 116326478A
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glabridin
glycyrrhiza glabra
medium
glabra
seedling
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CN116326478B (en
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王瑛
李勇青
马小玲
蒋宁馨
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South China Botanical Garden of CAS
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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
    • A01H4/002—Culture media for tissue culture
    • 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
    • A01H4/008—Methods for regeneration to complete plants

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Abstract

The invention provides a Glycyrrhiza glabra seedling culture medium and application thereof. The seed culture medium can be used for treating the glabra seed seedlings, can rapidly and effectively improve the glabridin content of the glabra roots, and especially can improve the glabridin content by more than 20 times when sodium bicarbonate alkali is used, thereby improving the effect obviously. The method for improving glabridin in the glabra provided by the invention has the advantages of simplicity, rapidness, high efficiency, low cost and the like, and has wide application space. The glabra licorice seedling culture medium is used for culturing the glabra licorice, and can obviously improve the glabridin content in the glabra licorice.

Description

Glycyrrhiza glabra seedling culture medium and application thereof
Technical Field
The invention belongs to the technical field of plant culture, and particularly relates to a glycyrrhiza glabra seedling culture medium and application thereof.
Background
Licorice is used as a traditional Chinese medicine with long history and plays an important role in treating diseases from ancient times to date. The main components of the liquorice comprise triterpenoid saponins (mainly glycyrrhizic acid), flavonoids, coumarins, alkaloids, polysaccharides, amino acids and the like, wherein the triterpenoid saponins (content of 4% -24%) and the flavonoids (content of 1% -5%) are main active components. Glabridin (Glabridin) is an isoflavone compound extracted from the rhizome of Glycyrrhiza glabra, and has a high content (about 11%) in total flavonoids of Glycyrrhiza glabra, and is a characteristic compound of Glycyrrhiza glabra. Glabridin has a wide range of pharmacological activities and is often added as a functional ingredient in a variety of high-end cosmetics, and is called "whitening gold" in the reputation of people. Therefore, the research on the glabridin has more practical application value. However, the biosynthetic pathways and molecular regulatory mechanisms of glabridin in glabridin are not clear.
The Chinese wild licorice is mainly distributed in the areas of the yellow river in the north provinces, such as Qinghai, inner Mongolia, gansu, xinjiang, ningxia, northeast and the like, and the Glycyrrhiza glabra is mainly distributed in the places with sufficient water sources in the south-north slopes of the Tianshan in Xinjiang. The distribution areas have sufficient illumination, winter cold and summer heat and large day and night temperature difference, so that the liquorice has the growth characteristics of light preference, cold resistance, heat resistance and salt and alkali resistance.
Therefore, the influence of accumulation of the glabridin is explored by regulating and controlling the growth factors of the liquorice, so that a reference can be provided for the utilization of the glabridin, and a certain foundation is laid for better market application of developing the liquorice.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the first aspect of the invention provides a glabra licorice seedling culture medium, and the culture medium can effectively improve the glabridin content in the glabra licorice.
In a second aspect, the invention provides a method for increasing glabridin in Glycyrrhiza glabra.
The third aspect of the invention provides an application of a Glycyrrhiza glabra seedling culture medium in Glycyrrhiza glabra cultivation.
According to a first aspect of the present invention, there is provided a Glycyrrhiza glabra seedling culture medium comprising at least one of sodium salt, methyl jasmonate (MeJA), 5-azacytidine (5-azaC).
In some embodiments of the invention, the sodium salt comprises sodium bicarbonate or sodium bicarbonate with NaCl, na 2 SO 4 Or Na (or) 2 CO 3 At least one of the combinations of the above.
In some embodiments of the invention, the sodium salt comprises sodium bicarbonate with NaCl, na 2 SO 4 、Na 2 CO 3 Is a combination of (a) and (b).
In some embodiments of the invention, the NaHCO 3 、NaCl、Na 2 SO 4 、Na 2 CO 3 The mass ratio of (2) is 9:1:1:9.
In some embodiments of the invention, when the medium contains sodium salt, the concentration of the sodium salt in the medium is 5 mM-30 mM, further 8 mM-20 mM, still further 8 mM-15 mM.
In some embodiments of the invention, when sodium bicarbonate is present in the medium, the concentration of sodium bicarbonate in the medium is 5 mM-20 mM, further 8 mM-16 mM, still further 10 mM-15 mM.
In some embodiments of the invention, when 5-azacytidine is contained in the medium, the concentration of 5-azacytidine in the medium is 10. Mu.M to 50. Mu.M, further 15. Mu.M to 40. Mu.M, still further 25. Mu.M to 35. Mu.M.
In some embodiments of the invention, when methyl jasmonate is present in the medium, the concentration of methyl jasmonate in the medium is 60. Mu.M to 120. Mu.M, further 80. Mu.M to 115. Mu.M, still further 90. Mu.M to 110. Mu.M.
In some embodiments of the invention, the Glycyrrhiza glabra seedling medium also includes MS medium.
In some preferred embodiments of the invention, the concentration of the MS medium is 4g/L to 5g/L.
In some preferred embodiments of the present invention, the Glycyrrhiza glabra seedling medium further comprises 15g/L to 25g/L sucrose and 0.4g/L to 0.6g/L morpholinoethanesulfonic acid.
In some preferred embodiments of the invention, the pH of the Glycyrrhiza glabra seedling medium is 5.7-6.0.
According to a second aspect of the present invention, there is provided a method for increasing glabridin in Glycyrrhiza glabra, comprising: culturing Glycyrrhiza glabra seed seedlings in the Glycyrrhiza glabra seedling culture medium according to the first aspect.
In some embodiments of the invention, the Glycyrrhiza glabra seed seedlings are those after pretreatment, the pretreatment being: soaking Glycyrrhiza glabra seeds in concentrated sulfuric acid, and washing with water until no concentrated sulfuric acid residue; then adding water for soaking, then sterilizing and washing; finally, the mixture is placed in sterile water for shake culture, and the just germinated glycyrrhiza glabra seedlings are obtained.
In some embodiments of the invention, the concentrated sulfuric acid soaking time is 15min to 30min.
In some embodiments of the invention, the soaking in water is for 2-3 hours.
In some embodiments of the invention, the disinfection includes, but is not limited to, disinfection with NaClO at a concentration of 2%.
In some embodiments of the invention, the disinfection time is 10min to 15min.
In some embodiments of the invention, the shake culture temperature is 25-30 ℃ and the rotation speed is 200-300 rpm; the time is 20-30 h.
In some embodiments of the invention, when the Glycyrrhiza glabra seedling medium contains sodium salt, the cultivation time is more than or equal to 24 hours, preferably 48 to 72 hours.
In some embodiments of the invention, when the glabra seedling medium contains methyl jasmonate, the cultivation time is more than or equal to 72 hours, preferably 72 to 120 hours.
In some embodiments of the invention, when the Glycyrrhiza glabra seedling medium contains 5-azacytidine, the cultivation time is greater than or equal to 120 hours, preferably 120 to 168 hours.
In some embodiments of the present invention, when the Glycyrrhiza glabra seedling medium contains more than one of the above-mentioned active ingredients (sodium salt, methyl jasmonate (MeJA), 5-azacytidine (5-azaC)), the time of the cultivation is based on the time of the cultivation required for the ingredient in which the time of the cultivation is the shortest.
According to a third aspect of the present invention, there is provided an application of the Glycyrrhiza glabra seedling culture medium according to the first aspect in Glycyrrhiza glabra cultivation.
In some embodiments of the invention, the glabra seed culture medium is used to increase glabridin content in glabra seed roots.
The beneficial effects of the invention are as follows:
1. the invention discovers for the first time that the glabra seed seedling is treated by the seed seedling culture medium, the glabra root glabridin content can be rapidly and effectively improved, and especially when sodium bicarbonate alkali is used, the glabridin content can be improved by more than 20 times, and the improvement effect is remarkable.
2. The method for improving glabridin in the glabra provided by the invention has the advantages of simplicity, rapidness, high efficiency, low cost and the like, and has wide application space.
3. The glabra licorice seedling culture medium is used for culturing the glabra licorice, and can obviously improve the glabridin content in the glabra licorice.
Drawings
The invention is further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a linear relationship between different concentrations of glabridin and peak area detected by HPLC.
FIG. 2A is a sample of the invention in example 1 containing 10mM NaHCO 3 The phenotype of the glycyrrhiza glabra seedlings cultured on the culture medium for 48 hours; b is the content detection result of the glabridin.
FIG. 3A is a sample of the present invention in example 1 containing 10mM complex salt (NaHCO 3 、NaCl、Na 2 SO 4 、Na 2 CO 3 ) The phenotype of the glycyrrhiza glabra seed seedlings is cultivated on a seedling culture medium for 48 hours; b is the content detection result of the glabridin.
FIG. 4A shows the phenotype of Glycyrrhiza glabra seedlings grown for 72h on seedling medium containing 100. Mu.M MeJA in example 1 of the present invention; b is the content detection result of the glabridin.
FIG. 5A is a phenotype of Glycyrrhiza glabra seedlings grown for 120h on a seedling medium containing 30. Mu.M 5-azaC in accordance with example 1 of the present invention; b is the content detection result of the glabridin.
FIG. 6A is a sample of the present invention in example 1 containing 10mM complex salt (NaHCO 3 、NaCl、Na 2 SO 4 、Na 2 CO 3 ) Phenotype of Glycyrrhiza glabra seed seedlings cultured for 120h on seedling medium of +100. Mu.M MeJA+30. Mu.M 5-azaC; b is the content detection result of the glabridin.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by a person skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention, and that conditions not mentioned in the embodiments are conventional conditions in the art. If not specified, the time period is 1 day, namely 24 hours, the time period is 2 days, namely 48 hours, and so on.
The materials used in the following examples were formulated as follows:
a basal medium comprising the following components: MS culture medium (Murashige & Skoog 15Basal Medium with Vitamins) with the final concentration of 4 g/L-5 g/L, sucrose with the final concentration of 15 g/L-20 g/L, MES (morpholinoethanesulfonic acid) with the final concentration of 0.4 g/L-0.6 g/L and the pH value of 5.7-6.0.
Containing 10mM NaHCO 3 The preparation of the glabra licorice seedling culture medium comprises the following steps: weighing 0.8401g NaHCO 3 Powder, added to 1L of the basic medium, and autoclaved.
The preparation of the 100mM methyl jasmonate (MeJA) mother liquor comprises the following steps: 2.3 mu L of MeJA stock solution is taken, diluted in 100 mu L of absolute ethanol and filtered and sterilized by a 0.22 mu m filter membrane.
The preparation of the Glycyrrhiza glabra seedling culture medium containing 100 mu M methyl jasmonate (MeJA) comprises the following steps: 1L of the basal medium was autoclaved and MeJA mother liquor was added at a final concentration of 100. Mu.M at a ratio of 1:1000 before cooling and solidification.
Containing 10mM complex salt (NaHCO) 3 、NaCl、Na 2 SO 4 、Na 2 CO 3 ) The preparation of the glabra licorice seedling culture medium comprises the following steps: weighing 0.26g NaCl,0.071g NaSO 4 ，0.42g NaHCO 3 ，0.477g Na 2 CO 3 Powder, added to 1L of the basic medium, and autoclaved.
The 30mM 5-azaC stock solution formulation included the following steps: 10mg of 5-azaC powder was weighed, dissolved in 1.365mL of pure water, and filtered through a 0.22 μm filter to be sterilized.
The preparation of the Glycyrrhiza glabra seedling culture medium containing 30 mu M5-azaC comprises the following steps: 1L of the basal medium was autoclaved and 5-azaC mother liquor was added at a final concentration of 30. Mu.M at a ratio of 1:1000 before cooling and solidification.
The preparation of the Glycyrrhiza glabra seedling culture medium containing 10mM complex salt and 100 mu M MeJA and 30 mu M5-azaC comprises the following steps: weighing 0.26g NaCl,0.071g NaSO 4 ，0.42g NaHCO 3 ，0.477g Na 2 CO 3 Powder was added to 1L of the above basal medium, autoclaved, and MeJA mother liquor and 5-azaC mother liquor were added at a final concentration of 100. Mu.M and 30. Mu.M, respectively, at a ratio of 1:1000 before cooling and solidification.
The following examples and comparative examples were obtained by the following methods:
placing the Glycyrrhiza glabra seeds into a 50mL centrifuge tube, adding 10mL concentrated sulfuric acid, and soaking for 20-30 min. The concentrated sulfuric acid is sucked out, and the pure water is quickly washed for 5 times, 15mL each time, until no concentrated sulfuric acid remains. Soaking in pure water for 2h, and sterilizing with 20mL of 2% NaClO in an ultra clean bench for 15min. After pouring out NaClO, the mixture was rinsed with 15mL of sterile pure water for 10min, and then sterilized with 20mL of 2% NaClO for 5min. Then, the mixture was washed with 15mL of sterilized pure water 5 times, and allowed to stand for 10 minutes each time. After cleaning, 20mL of sterile pure water is added, shake culture is carried out for 24 hours at 28 ℃ and 200rpm/min, and the glabra seed starts to germinate, thus obtaining the freshly germinated glabra seed seedling.
Example 1
After the glabra seed seedlings sprout on the basal medium for 2 days, transferring the glabra seed seedlings to a new basal medium for vertical culture, recovering for 5 days, and transferring the glabra seed seedlings to the culture medium containing NaHCO respectively 3 (final concentration 10 mM) on seedling medium for 2 days, and collecting root of Glycyrrhiza glabra seed seedling.
Grinding the root of the Glycyrrhiza glabra seed seedling obtained by the culture with liquid nitrogen at the frequency of 45Hz for 35s for 1 time, and freeze-drying at-50deg.C for 48h to obtain the sample.
Adding 1mL of absolute methanol into the sample, performing ultrasonic treatment at 25 ℃ for 30min, preparing a new centrifuge tube, marking, centrifuging after ultrasonic treatment, and sucking the supernatant into the new centrifuge tube by using a pipette; adding 1mL of anhydrous methanol into the sample again, performing ultrasonic treatment for 30min, combining the filtrates, evaporating to dryness and concentrating to obtain the extract.
Example 2
After sprouting for 2 days on the basal medium, the glabra seedlings are transferred to a new basal medium for vertical culture, recovered for 5 days, transferred to the seedling culture medium containing compound salt (the sum of the final concentration of each component is 10 mM) for 2 days, and then the roots of the glabra seedlings are collected.
Grinding the root of the Glycyrrhiza glabra seed seedling obtained by the culture with liquid nitrogen at the frequency of 45Hz for 35s for 1 time, and freeze-drying at-50deg.C for 48h to obtain the sample.
Adding 1mL of absolute methanol into the sample, performing ultrasonic treatment at 25 ℃ for 30min, preparing a new centrifuge tube, marking, centrifuging after ultrasonic treatment, and sucking the supernatant into the new centrifuge tube by using a pipette; adding 1mL of anhydrous methanol into the sample again, performing ultrasonic treatment for 30min, combining the filtrates, evaporating to dryness and concentrating to obtain the extract.
Example 3
After sprouting for 2 days on the basal medium, the glabra seedlings are transferred to a new basal medium for vertical culture, recovered for 5 days, transferred to the seedling culture medium containing methyl jasmonate (with the final concentration of 100 mu M) for 3 days, and then the roots of the glabra seedlings are collected.
Grinding the root of the Glycyrrhiza glabra seed seedling obtained by the culture with liquid nitrogen at the frequency of 45Hz for 35s for 1 time, and freeze-drying at-50deg.C for 48h to obtain the sample.
Adding 1mL of absolute methanol into the sample, performing ultrasonic treatment at 25 ℃ for 30min, preparing a new centrifuge tube, marking, centrifuging after ultrasonic treatment, and sucking the supernatant into the new centrifuge tube by using a pipette; adding 1mL of anhydrous methanol into the sample again, performing ultrasonic treatment for 30min, combining the filtrates, evaporating to dryness and concentrating to obtain the extract.
Example 4
After the Glycyrrhiza glabra seed seedlings sprout on the culture medium for 2 days, transferring to a new culture medium for vertical culture, recovering for 5 days, transferring to the culture medium containing 5-azaC (30 mu M final concentration) for 5 days, and collecting the roots of the Glycyrrhiza glabra seed seedlings.
Grinding the root of the Glycyrrhiza glabra seed seedling obtained by the culture with liquid nitrogen at the frequency of 45Hz for 35s for 1 time, and freeze-drying at-50deg.C for 48h to obtain the sample.
Adding 1mL of absolute methanol into the sample, performing ultrasonic treatment at 25 ℃ for 30min, preparing a new centrifuge tube, marking, centrifuging after ultrasonic treatment, and sucking the supernatant into the new centrifuge tube by using a pipette; adding 1mL of anhydrous methanol into the sample again, performing ultrasonic treatment for 30min, combining the filtrates, evaporating to dryness and concentrating to obtain the extract.
Example 5
After sprouting for 2 days on basal medium, the Glycyrrhiza glabra seedlings are transferred to new basal medium for vertical culture and recovered for 5 days, then transferred to seedling culture medium containing compound salt (final concentration is 10 mM), methyl jasmonate (final concentration is 100. Mu.M) and 5-azaC (final concentration is 30. Mu.M) for 5 days, and then the roots of Glycyrrhiza glabra seedlings are collected.
Grinding the root of the Glycyrrhiza glabra seed seedling obtained by the culture with liquid nitrogen at the frequency of 45Hz for 35s for 1 time, and freeze-drying at-50deg.C for 48h to obtain the sample.
Adding 1mL of absolute methanol into the sample, performing ultrasonic treatment at 25 ℃ for 30min, preparing a new centrifuge tube, marking, centrifuging after ultrasonic treatment, and sucking the supernatant into the new centrifuge tube by using a pipette; adding 1mL of anhydrous methanol into the sample again, performing ultrasonic treatment for 30min, combining the filtrates, evaporating to dryness and concentrating to obtain the extract.
Comparative example 1
After the Glycyrrhiza glabra seed seedlings sprout on the basal medium for 2 days, the Glycyrrhiza glabra seed seedlings are transferred to a new basal medium for vertical culture, recovered for 5 days, transferred to the basal medium for 2 days, and then the roots of the Glycyrrhiza glabra seed seedlings are collected as a Control group (Control) of example 1.
Grinding the root of the Glycyrrhiza glabra seed seedling obtained by the culture with liquid nitrogen at the frequency of 45Hz for 35s for 1 time, and freeze-drying at-50deg.C for 48h to obtain the sample.
Adding 1mL of absolute methanol into the sample, performing ultrasonic treatment at 25 ℃ for 30min, preparing a new centrifuge tube, marking, centrifuging after ultrasonic treatment, and sucking the supernatant into the new centrifuge tube by using a pipette; adding 1mL of anhydrous methanol into the sample again, performing ultrasonic treatment for 30min, combining the filtrates, evaporating to dryness and concentrating to obtain the extract.
Comparative example 2
After the Glycyrrhiza glabra seed seedlings sprout on the basal medium for 2 days, the Glycyrrhiza glabra seed seedlings are transferred to a new basal medium for vertical culture, recovered for 5 days, transferred to the basal medium for 2 days, and then the roots of the Glycyrrhiza glabra seed seedlings are collected as a Control group (Control) of example 2.
Grinding the root of the Glycyrrhiza glabra seed seedling obtained by the culture with liquid nitrogen at the frequency of 45Hz for 35s for 1 time, and freeze-drying at-50deg.C for 48h to obtain the sample.
Adding 1mL of absolute methanol into the sample, performing ultrasonic treatment at 25 ℃ for 30min, preparing a new centrifuge tube, marking, centrifuging after ultrasonic treatment, and sucking the supernatant into the new centrifuge tube by using a pipette; adding 1mL of anhydrous methanol into the sample again, performing ultrasonic treatment for 30min, combining the filtrates, evaporating to dryness and concentrating to obtain the extract.
Comparative example 3
After the Glycyrrhiza glabra seed seedlings sprout on the basal medium for 2 days, the Glycyrrhiza glabra seed seedlings are transferred to a new basal medium for vertical culture, recovered for 5 days, transferred to the basal medium for 3 days, and then the roots of the Glycyrrhiza glabra seed seedlings are collected as a Control group (Control) of example 3.
Grinding the root of the Glycyrrhiza glabra seed seedling obtained by the culture with liquid nitrogen at the frequency of 45Hz for 35s for 1 time, and freeze-drying at-50deg.C for 48h to obtain the sample.
Adding 1mL of absolute methanol into the sample, performing ultrasonic treatment at 25 ℃ for 30min, preparing a new centrifuge tube, marking, centrifuging after ultrasonic treatment, and sucking the supernatant into the new centrifuge tube by using a pipette; adding 1mL of anhydrous methanol into the sample again, performing ultrasonic treatment for 30min, combining the filtrates, evaporating to dryness and concentrating to obtain the extract.
Comparative example 4
After the Glycyrrhiza glabra seed seedlings sprout on the basal medium for 2 days, the Glycyrrhiza glabra seed seedlings are transferred to a new basal medium for vertical culture, recovered for 5 days, transferred to the basal medium for 5 days, and then the roots of the Glycyrrhiza glabra seed seedlings are collected as a Control group (Control) of example 4.
Grinding the root of the Glycyrrhiza glabra seed seedling obtained by the culture with liquid nitrogen at the frequency of 45Hz for 35s for 1 time, and freeze-drying at-50deg.C for 48h to obtain the sample.
Adding 1mL of absolute methanol into the sample, performing ultrasonic treatment at 25 ℃ for 30min, preparing a new centrifuge tube, marking, centrifuging after ultrasonic treatment, and sucking the supernatant into the new centrifuge tube by using a pipette; adding 1mL of anhydrous methanol into the sample again, performing ultrasonic treatment for 30min, combining the filtrates, evaporating to dryness and concentrating to obtain the extract.
Comparative example 5
After the Glycyrrhiza glabra seed seedlings sprout on the basal medium for 2 days, the Glycyrrhiza glabra seed seedlings are transferred to a new basal medium for vertical culture, recovered for 5 days, transferred to the basal medium for 5 days, and then the roots of the Glycyrrhiza glabra seed seedlings are collected as a Control group (Control) of example 4.
Grinding the root of the Glycyrrhiza glabra seed seedling obtained by the culture with liquid nitrogen at the frequency of 45Hz for 35s for 1 time, and freeze-drying at-50deg.C for 48h to obtain the sample.
Adding 1mL of absolute methanol into the sample, performing ultrasonic treatment at 25 ℃ for 30min, preparing a new centrifuge tube, marking, centrifuging after ultrasonic treatment, and sucking the supernatant into the new centrifuge tube by using a pipette; adding 1mL of anhydrous methanol into the sample again, performing ultrasonic treatment for 30min, combining the filtrates, evaporating to dryness and concentrating to obtain the extract.
Test examples
The extracts obtained in example 1 and comparative example 1, example 2 and comparative example 2, example 5 and comparative example 5 were subjected to glabridin content test, 200. Mu.L of anhydrous methanol was respectively sucked up to resuspend the above extracts, the obtained extracts were respectively filtered with 0.22 μm filter membranes, and the filtrates were respectively collected as sample solutions to be tested for sample introduction, each group was repeated 3 times.
The glabridin content test was performed on the extracts obtained in example 3 and comparative example 3, example 4 and comparative example 4, respectively, using HPLC, 300 μl of anhydrous methanol was respectively sucked up to resuspend the above extracts, the obtained extracts were respectively filtered with 0.22 μm filter membranes, and the filtrates were respectively collected as sample solutions to be tested for sample introduction, each group was repeated 3 times.
(1) Chromatographic system: by C 18 Column (150 mm. Times.4.6 mm,5 μm, shimadzu, kyoto, japan), column temperature 40 ℃, flow rate 1mL/min, sample injection volume 10. Mu.L; mobile phase a: acetonitrile, mobile phase B:0.1% (V/V) aqueous formic acid;
gradient elution (in volume fraction of each mobile phase): t=0 min,30% a,70% b;
t＝25min，55％A，45％B；
t＝27min，95％A，5％B；
t＝30min，95％A，5％B；
t＝31min，30％A，70％B；
t＝35min，30％A，70％B；
the detection wavelength of glabridin is 280nm, and the number of theoretical plates is calculated according to glabridin peak, and should be no less than 8000.
(2) Calculating glabridin content: detecting peak areas on HPLC by using glabridin with different concentrations, establishing a standard curve between the concentration and the peak areas, wherein good linearity is realized between the quality concentration of the standard and the absorption value intensity, and a calculation formula for obtaining the concentration according to the standard curve between the glabridin concentration and the peak areas is as follows: y=25992x+314.91. The linear relationship between different concentrations of glabridin and peak area detected by HPLC is shown in FIG. 1, and it can be seen that there is good linearity between the concentration of glabridin and the intensity of absorption value.
Wherein example 1 is performed using a solution containing 10mM NaHCO 3 Treating samples of Glycyrrhiza glabra seed seedlings for 2 days with seedling culture mediumThe detection result of the glabridin is: peak areas were 29848, 15767, 18366, respectively. Calculated to be 10mM NaHCO using the above formula 3 The concentrations of glabridin in the treated glabra seed seedling extract are respectively as follows: 1.136. Mu.g/mL, 0.594. Mu.g/mL, 0.694. Mu.g/mL. Wherein the dry weight of the samples is: 6.0mg, 3.3mg, 4.2mg, and finally dissolved in 200. Mu.L of anhydrous methanol. From this the NaHCO was calculated according to the formula "glabridin content= (glabridin concentration x solution volume)/dry weight 3 The contents of glabridin in the treated glabra seedlings are respectively as follows: 37.87ng/mg DW, 36.03ng/mg DW, 33.07ng/mg DW.
Example 2 glabridin in samples of glabridin seedlings treated with seedling medium containing 10mM complex salt for 2 days gave the following results: peak areas are 1682, 4371, 11234, respectively. The concentrations of glabridin in the 10mM complex salt treated glabra seed seedling extract were calculated by using the above formula, respectively: 0.052. Mu.g/mL, 0.156. Mu.g/mL, 0.420. Mu.g/mL. Wherein the dry weight of the samples is: 11.6mg, 12.4mg, 21.2mg, were finally dissolved in 200. Mu.L of anhydrous methanol. From this, the content of glabridin in the glabridin seedlings treated with the compound salt was calculated according to the formula "content of glabridin= (glabridin concentration of solution volume)/dry weight" as follows: 0.91ng/mg DW, 2.52ng/mg DW, 3.96ng/mg DW.
Example 3 glabridin in samples of glabra seedlings treated with 100 μm MeJA seedling medium for 3 days was detected as follows: peak areas were 7747, 8740, 7031, respectively. The concentrations of glabridin in the 100 μm MeJA treated glabra seed seedling extract were calculated by using the above formula, respectively: 0.286. Mu.g/mL, 0.324. Mu.g/mL, 0.258. Mu.g/mL. Wherein the dry weight of the samples is respectively: 20.5mg, 17.4mg, 22.4mg, and finally dissolved in 300. Mu.L of anhydrous methanol. From this, the content of glabridin in MeJA-treated glabridin seedlings was calculated according to the formula "glabridin content= (glabridin concentration x solution volume)/dry weight" as follows: 4.18ng/mg DW, 5.59ng/mg DW, 3.46ng/mg DW.
Example 4 glabridin in samples of glabridin seedlings of Glycyrrhiza glabra seeds treated with seedling medium containing 30. Mu.M 5-azaC for 5 days was detected as follows: peak areas are 2013, 1564, 1244, respectively. The concentrations of glabridin in the 30 μm5-azaC treated glabra seed seedling extract were calculated using the above formula, respectively: 0.065 μg/mL, 0.048 μg/mL, 0.036 μg/mL. Wherein the dry weight of the samples is respectively: 15.1mg, 20.3mg, 11.4mg, were finally dissolved in 300. Mu.L of anhydrous methanol. From this, the content of glabridin in the 5-azaC treated glabra seedlings was calculated according to the formula "glabridin content= (glabridin concentration x solution volume)/dry weight" as follows: 1.30ng/mg DW, 0.71ng/mg DW, 0.94ng/mg DW.
Example 5 glabridin in samples of glabridin seedlings treated with seedling medium containing 10mM complex salt + 100. Mu.M methyl jasmonate + 30. Mu.M 5-azaC for 5 days was detected as follows: peak areas were 29589, 8972, 68701, respectively. The concentrations of glabridin in the 10mM complex salt + 100. Mu.M methyl jasmonate + 30. Mu.M 5-azaC treated glabra seed seedling extract were calculated using the above formula, respectively: 1.126. Mu.g/mL, 0.333. Mu.g/mL, 2.631. Mu.g/mL. Wherein the dry weight of the samples is respectively: 19.1mg, 10.3mg, 19.4mg, were finally dissolved in 200. Mu.L of anhydrous methanol. From this, the compound salt + methyl jasmonate +5-azaC treated glabridin seed seedlings were calculated according to the formula "glabridin content= (glabridin concentration x solution volume)/dry weight" as follows: 11.79ng/mg DW, 6.47ng/mg DW, 24.34ng/mg DW.
As shown in FIG. 2, panel A shows the phenotype of the glabridin seedlings of comparative example 1 and example 1 at the time of harvesting, and Panel B shows the content of glabridin seedlings of comparative example 1 and example 1 (after averaging) by HPLC. And unused NaHCO 3 Treated comparative example 1 contains 10mM NaHCO 3 The seedling culture medium is used for treating the glabra seed seedlings for 2 days, and obvious glabridin accumulation exists. In comparative example 1, however, no glabridin was detected, indicating that accumulation of glabridin was too small and the content was below the detection limit.
As shown in FIG. 3, panel A shows the phenotype of the glabra seedlings of comparative example 2 and example 2 at the time of harvesting, and Panel B shows the content of glabridin in the seedlings extract of comparative example 2 and example 2 (after averaging). Compared with comparative example 2, which did not use the complex salt treatment, the seedling medium containing 10mM complex salt treated Glycyrrhiza glabra seed seedlings for 2 days, and there was a significant accumulation of glabridin. In comparative example 2, however, no glabridin was detected, indicating that the accumulation of glabridin was too small and the content was below the detection limit.
As shown in FIG. 4, panel A shows the phenotype of the glabridin seedlings of comparative example 3 and example 3 at the time of harvesting, and Panel B shows the content of glabridin in the glabridin the seedlings extract of comparative example 2 and example 2 (after averaging). Compared with comparative example 3, which was not treated with MeJA, there was a significant accumulation of glabridin when the glabridin seed was treated with 100 μm MeJA-containing seedling medium for 3 days. In comparative example 3, however, no glabridin was detected, indicating that the accumulation of glabridin was too small and the content was below the detection limit.
As shown in FIG. 5, panel A shows the phenotype of the glabra seedlings of comparative example 4 and example 4 at the time of harvesting, and Panel B shows the content of glabridin in the seedlings extract of comparative example 4 and example 4 (after averaging). There was a significant accumulation of glabridin in the seedlings of Glycyrrhiza glabra seed treated with 30. Mu.M of 5-azaC in seedling medium for 5 days, as compared to comparative example 4 without 5-azaC treatment. In contrast, no glabridin was detected in comparative example 4, which showed that accumulation of glabridin was too little and the content was below the detection limit.
As shown in FIG. 6, panel A shows the phenotype of the glabridin seedlings of comparative example 5 and example 5 at the time of harvesting, and Panel B shows the content of glabridin in the glabridin the seedlings of comparative example 5 and example 5 (after averaging). Compared with comparative example 5 which did not use the complex salt + methyl jasmonate +5-azaC treatment, there was a significant accumulation of glabridin in the seedlings treated with seedling medium containing 10mM complex salt + 100. Mu.M methyl jasmonate + 30. Mu.M 5-azaC for 5 days. In contrast, no glabridin was detected in comparative example 4, which showed that accumulation of glabridin was too little and the content was below the detection limit.
From the aspects of wild growth environment and hormone action of the glycyrrhiza glabra, the synthesis and accumulation of secondary metabolites in the glycyrrhiza glabra can be promoted by adopting proper growth factor stimulation. The results show that the use of the glabra seed culture medium disclosed by the invention can promote the response of glabra seed seedlings to the stimulating factors, and further promote the gene expression related to the accumulation of glabridin, so that the accumulation of glabridin is rapidly and effectively promoted.
While the embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.

Claims (10)

1. A glycyrrhiza glabra seedling culture medium, which is characterized by comprising at least one of sodium salt, methyl jasmonate and 5-azacytidine.
2. The Glycyrrhiza glabra seedling culture medium of claim 1, wherein the sodium salt comprises sodium bicarbonate or sodium bicarbonate and NaCl, na 2 SO 4 Or Na (or) 2 CO 3 At least one of the combinations of the above.
3. The Glycyrrhiza glabra seedling culture medium of claim 2, wherein the sodium salt comprises sodium bicarbonate, naCl and Na 2 SO 4 、Na 2 CO 3 Is a combination of (a) and (b).
4. A Glycyrrhiza glabra seedling medium as claimed in claim 3, wherein the NaHCO 3 、NaCl、Na 2 SO 4 、Na 2 CO 3 The mass ratio of (2) is 1:9:1:9.
5. The Glycyrrhiza glabra seedling medium of claim 2, wherein when the medium contains sodium salt, the concentration of the sodium salt in the medium is 5 mM-30 mM.
6. The Glycyrrhiza glabra seedling culture medium of claim 5, wherein when the medium contains sodium bicarbonate, the concentration of sodium bicarbonate in the medium is 5 mM-20 mM.
7. The Glycyrrhiza glabra seedling medium of claim 1, wherein when the medium contains 5-azacytidine, the concentration of 5-azacytidine in the medium is 10 μm to 50 μm.
8. The Glycyrrhiza glabra seedling medium of claim 1, wherein when the medium contains methyl jasmonate, the concentration of the methyl jasmonate in the medium is 60 μm to 120 μm.
9. A method for increasing glabridin in glycyrrhiza glabra, which is characterized in that a glycyrrhiza glabra seed seedling is placed in the glabra seedling culture medium according to any one of claims 1-8 for culture.
10. Use of the Glycyrrhiza glabra seedling medium of any one of claims 1-8 in Glycyrrhiza glabra cultivation.
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