CN112753410A - Method for improving alkaloid content in lycoris by using exogenous substances - Google Patents
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Abstract
The invention provides a method for improving alkaloid content in lycoris by using exogenous substances, and relates to the field of plant growth and secondary metabolite accumulation regulation. According to the invention, exogenous substances 4' -O-methyl norallicin and/or methyl jasmonate are used for treating 3-month-1-year-old tissue culture seedlings of the lycoris longiradiata, so that the alkaloid contents of galanthamine, lycorine and lycoramine which have important medicinal values in the lycoris longiradiata are effectively increased.
Description
Technical Field
The invention relates to the technical field of plant growth and secondary metabolite accumulation regulation, in particular to a method for improving alkaloid content in lycoris by using exogenous substances.
Background
Lycoris longiradiata (Lycoris longitaba) is a bulbous herbaceous plant of Amaryllidaceae, has high ornamental value, also has medicinal function, and is rich in alkaloid components unique to various Amaryllidaceae plants. Lycorine (LYC) can induce apoptosis of cancer cell line HL-60, and Galantamine (GAL) and Lycoramine (LYCM) are one of the first choice drugs for clinical treatment of Alzheimer disease. However, the content of the three alkaloids in wild lycoris plants is low (only a few parts per million to a few parts per million), and the three alkaloids are influenced by provenance, development period and environmental conditions, so that the yield is low, and the market demand can not be met. The chemical synthesis has high cost and complicated steps. The whole plant of the lycoris plant contains alkaloid, and the lycoris alkaloid is mainly extracted from natural plants at the present stage.
The elicitor belongs to a unique elicitor, can generate defense reaction in plants, has the characteristics of selection, rapidness, high specificity and the like when inducing the expression of specific genes, and can increase the yield of specific secondary metabolites of the plants. The elicitor is widely applied to the research field of cell secondary metabolism, can promote the accumulation of medicinal components in the cell culture of medicinal plants, and is an effective measure. In recent years, elicitors have been widely used to induce the synthesis of natural secondary metabolites such as phenols, alkaloids, terpenes, and flavonoids. The precursor is formed in the metabolic process, and the small molecular substance generated in a secondary metabolic mode is called as a precursor substance and has an effect on the growth of plant tissues and cells and the synthesis of secondary metabolites, so that the accumulation of the yield of the metabolites is promoted.
Under the condition of artificial planting or natural growth, the growth of the lycoris longituba can be greatly influenced by factors such as plant diseases and insect pests, climate and the like, so that the alkaloid content is reduced, the effective yield is reduced, and the shortage of supply in the traditional Chinese medicine market is further forced. However, with the development of tissue culture technology, the accumulation of secondary metabolites of medicinal plants can be effectively promoted, so that the alkaloid content is increased, and the problems of plant diseases and insect pests, climatic conditions, seasonal conditions and the like can be solved, so that the tissue culture technology is one of effective alternative ways. However, the current research on the growth and alkaloid accumulation of lycoris longituba by precursor substances and elicitors is relatively lacking, so that a method for inducing the alkaloid accumulation of lycoris longituba needs to be researched, so that the alkaloid content in lycoris longituba can be increased.
Disclosure of Invention
The invention aims to provide a method for improving alkaloid content in lycoris radiata by utilizing a foreign substance, and the method is used for researching the influence of different adding concentrations and culture time on the growth and alkaloid accumulation of lycoris radiata by adding 4' -O-methyl norrhipid and/or methyl jasmonate into a lycoris radiata culture system. The contents of galanthamine, lycorine and lycoramine in the lycoris radiata treated by 4' -O-methyl norsolilolidine and/or methyl jasmonate are effectively improved.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for improving alkaloid content in lycoris by using exogenous substances, which comprises the following steps:
treating 3-1-year-old lycoris radiata tissue culture seedlings by using exogenous substances, wherein the exogenous substances are 4' -O-methyl noralothin and/or methyl jasmonate.
In the invention, the effective treatment concentration of the exogenous substance 4' -O-methyl norfarringidine is 0.1-0.2 g/L.
In the invention, the effective treatment concentration of the exogenous methyl jasmonate is 75-150 mu mol/L.
In the invention, the method for treating the tissue culture seedling by the exogenous substance is to place the tissue culture seedling on a liquid culture medium for continuous shake culture.
In the invention, the liquid culture medium is MS +6-BA 2.5-3.5 mg/L + NAA 0.8-1.5 mg/L + sucrose 25-35 g/L.
In the invention, the oscillation processing time is 7-30 d.
In the invention, the speed of the oscillation treatment is 100-120 rpm/min.
The culture method of the tissue culture seedling comprises the steps of inoculating disinfected lycoris radiata seeds on a culture medium to induce protogenic bulblets, cutting the protogenic bulblets into small pieces, inoculating the small pieces of protogenic bulblets on the culture medium to induce adventitious buds, inoculating the induced adventitious buds on the culture medium to proliferate the adventitious buds, inoculating the proliferated adventitious buds on the culture medium to perform expansion culture, and obtaining the tissue culture seedling after the expansion culture.
In the invention, the culture medium for performing the expansion culture is MS + 4.5-5.8 mg/L6-BA + 1-2 mg/L NAA + 0.5-2 mg/L CPPU + 25-35 g/L sucrose + 7-9 g/L agar.
In the invention, the culture temperature of the expansion culture is 22-24 ℃, and the illumination intensity is 40-55 mu mol/m2/s。
The invention has the following beneficial effects:
1. according to the invention, the tissue culture seedling of the Lycoris longituba is treated by using the precursor substance of the exogenous substance 4' -O-methyl norrhaponticum carteri (MN), so that the content of three alkaloids with important medicinal values in the Lycoris longituba is effectively improved, the galanthamine content is improved by 1.89 times compared with the contrast, the lycorine content is improved by 2.03 times compared with the contrast, and the lycoramine content is improved by 2.01 times compared with the contrast.
2. According to the invention, the tissue culture seedling of the lycoris longituba is treated by using the exogenous source methyl jasmonate (MeJA) inducer, so that the content of three alkaloids with important medicinal values in the lycoris longituba is effectively increased, the galanthamine content is increased by 1.05-2.70 times compared with the control, the lycorine content is increased by 2.01 times compared with the control, and the lycoramine content is increased by 1.56-2.85 times compared with the control.
3. The method does not need higher early investment required by improving the alkaloid content by utilizing biological technologies such as genetic engineering and the like, is simple to operate, is beneficial to shortening the production time of the lycoris plant alkaloid and reducing the cost, and has important significance for large-scale production of the anticancer alkaloid substances.
Drawings
FIG. 1 is a graph showing the detection results of galanthamine content of Lycoris longituba seedlings after being treated with MeJA of different concentrations for 7-28 days;
FIG. 2 is a graph showing the results of measuring the content of lycoramine after the lycoris longiradiata seedlings are treated by MeJA with different concentrations for 7-28 days;
FIG. 3 is a graph showing the results of measuring lycorine content of Lycoris longiradiata seedlings after MeJA treatment at different concentrations for 7-28 days.
Detailed Description
The invention provides a method for improving alkaloid content in lycoris by using exogenous substances, which comprises the following specific steps: treating 3-1-year-old lycoris radiata tissue culture seedlings by using exogenous substances, wherein the exogenous substances are 4' -O-methyl noralothin and/or methyl jasmonate. Experiments show that the alkaloid content in the lycoris radiata tissue culture seedlings can be greatly improved by selecting and inducing the tissue culture seedlings growing for 3 months to 1 year in expansion culture on the basis. In an embodiment of the present invention, the Lycoris radiata seeds and seedlings are from Lycoris radiata germplasm resource nursery of shanghai city academy of agricultural sciences, and the Lycoris radiata is longitauba. In the invention, the exogenous substance 4' -O-methyl norfarazid is purchased from Shanghai spectral Xin chemical technology Co., Ltd; the exogenous methyl jasmonate was purchased from Merck life sciences.
In the invention, the effective treatment concentration of the exogenous substance 4' -O-methyl norfarringidine is 0.1-0.2 g/L.
In the invention, the effective treatment concentration of the exogenous methyl jasmonate is 75-150 mu mol/L.
In the invention, the method for treating the tissue culture seedling by the exogenous substance is to place the tissue culture seedling on a liquid culture medium for continuous shake culture.
In the invention, the liquid culture medium is MS +6-BA 2.5-3.5 mg/L + NAA 0.8-1.5 mg/L + sucrose 25-35 g/L. The raw materials for the liquid medium described in the present invention were purchased from Merck Life sciences.
In the invention, the oscillation processing time is 7-30 d.
In the invention, the speed of the oscillation treatment is 100-120 rpm/min.
The culture method of the tissue culture seedling comprises the steps of inoculating disinfected lycoris radiata seeds on a culture medium to induce protogenic bulblets, cutting the protogenic bulblets into small pieces, inoculating the small pieces of protogenic bulblets on the culture medium to induce adventitious buds, inoculating the induced adventitious buds on the culture medium to proliferate the adventitious buds, inoculating the proliferated adventitious buds on the culture medium to perform expansion culture, and obtaining the tissue culture seedling after the expansion culture. In the specific embodiment of the invention, the seeds of the lycoris longituba are inoculated to MS +1 mg/L6-BA +0.2mg/L NAA +30g/L sucrose +8g/L agar after being sterilized, and the primary bulblet is induced on a culture medium with pH of 5.8; cutting the primary bulblet into 0.5cm small pieces, inoculating to MS +5.0 mg/L6-BA +0.5mg/L NAA +1.0mg/L CPPU +0.5mg/L TDZ +30g/L sucrose +8g/L agar, and inducing adventitious bud on a culture medium with pH of 5.8; and (3) inoculating the adventitious bud obtained by induction on a culture medium of MS +5.0 mg/L6-BA +1.5mg/L NAA +30g/L sucrose +8g/L agar and pH5.8 for adventitious bud proliferation, and performing expansion culture on the adventitious bud after proliferation culture on the culture medium for 3 months-1 year to obtain a tissue culture seedling.
In the invention, the culture medium for performing the expansion culture is MS + 4.5-5.8 mg/L6-BA + 1-2 mg/L NAA + 0.5-2 mg/L CPPU + 25-35 g/L sucrose + 7-9 g/L agar. Tests show that each culture medium used in the invention is also beneficial to increasing the content of three alkaloids in the lycoris. The raw materials for each of the media described in the present invention were purchased from Merck Life sciences.
In the invention, the culture temperature of the expansion culture is 22-24 ℃, and the illumination intensity is 40-55 mu mol/m2/s。
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The seed and seedling of Lycoris radiata in the following examples are from Lycoris radiata germplasm resource garden of Shanghai city academy of agricultural sciences, and the variety is Lycoris longituba (Lycoris longituba). Other Amaryllidaceae species may also be used, as the present invention is not limited in this regard.
Example 1
Selecting 3-month-long-tube lycoris radiata tissue culture seedlings with uniform growth state, inoculating the tissue culture seedlings on a liquid culture medium of MS +6-BA 3.0mg/L + NAA 1mg/L +30g/L sucrose, and subpackaging each bottle with 20ml of the culture medium. Adding 0.22 μm filter-sterilized 4' -O-Methyl Norcantharidin (MN) solution into culture medium, setting final concentrations to be 0.1g/L MN (T1) and 0.2g/L MN (T2), inoculating 2g Bulbus Lycoridis Radiatae tissue culture seedling to each bottle of liquid culture medium, inoculating 3 bottles of liquid culture medium for each different treatment, repeating for 3 times, and culturing at 23 deg.C and 50 μmol/m2Illuminating for 12 hours under the illumination intensity/s, and shakingThe oscillation frequency is 100 r/min. And taking out after culturing for 7d, 14d and 30d by using a liquid culture medium.
Example 2
Selecting annual tissue culture seedlings of lycoris longituba with uniform growth state, inoculating the annual tissue culture seedlings to a liquid culture medium of MS +6-BA 3.0mg/L + NAA 1mg/L +30g/L sucrose, and subpackaging each bottle with 20ml of the culture medium. Adding 0.22 μm filter-sterilized 4' -O-Methyl Norcantharidin (MN) solution into culture medium, setting 3 treatments to make final concentrations respectively 0g/L (T0), 0.1g/L MN (T1), 0.2g/L MN (T2), inoculating 2g long tube Bulbus Lycoridis Radiatae tissue culture seedling to each bottle of liquid culture medium, inoculating 3 bottles of liquid culture medium for each different treatment, setting 3 times, and repeating at 25 deg.C and 50 μmol/m2The light intensity is changed into the light intensity of 110r/min for 12 hours. Culturing in liquid culture medium for 7d, 14d and 30d, taking out, and determining the content of three alkaloids including galanthamine, lycoramine and lycorine by chromatograph connected with two-stage mass spectrometer in series.
The results of the measurements are shown in tables 1-3, where Galantamine (GAL), Lycorine (LYC) and Lycoramine (LYCM) were highest at 1.89 times, 2.03 times and 2.01 times higher than CK treatment (0g/L (T0)) for 7 days under T1 treatment. It was thus found that T1 treatment for 7 days was able to promote accumulation of GAL, LYC and LYCM in Lycoris longiradiata seedlings to varying degrees.
TABLE 1 accumulation of galanthamine in Lycoris radiata tissue culture seedlings (μ g. g)-1DW)
TABLE 2 accumulation of lycoramine in Lycoris longituba tissue culture seedlings (μ g. g)-1DW)
TABLE 3 accumulation of lycorine in Lycoris longituba tissue culture seedlings (μ g. g)-1DW)
Example 3
Selecting 1-year-old tissue culture seedlings of lycoris longituba with uniform growth state, inoculating the tissue culture seedlings on a liquid culture medium of MS +6-BA 3.0mg/L + NAA 1mg/L +30g/L sucrose, and subpackaging each bottle with 20ml of the culture medium. Adding 0.22 μm filter-sterilized methyl jasmonate (MeJA) solution into culture medium, setting final concentrations to be 75 μmol/L MeJA (MJ-75), 150 μmol/L MeJA (MJ-150) and 300 μmol/L MeJA (MJ-300), respectively, inoculating 2g long-tube lycoris tissue culture seedling to each bottle of liquid culture medium, inoculating 3 bottles of liquid culture medium for each different treatment, setting for 3 times, and culturing at 27 deg.C and 50 μmol/m2The light intensity is changed into light intensity of 120r/min for 12 hours. Culturing in liquid culture medium for 7d, 14d and 30d, taking out, and determining the content of three alkaloids including galanthamine, lycoramine and lycorine by chromatograph connected with two-stage mass spectrometer in series.
Example 4
Selecting 3-month-long-tube lycoris radiata tissue culture seedlings with uniform growth state, inoculating the tissue culture seedlings on a liquid culture medium of MS +6-BA 3.0mg/L + NAA 1mg/L +30g/L sucrose, and subpackaging each bottle with 20ml of the culture medium. Adding 0.22 μm filter-sterilized methyl jasmonate (MeJA) solution into culture medium, setting 4 treatments to final concentrations of 0 μmol/L (MJ-0), 75 μmol/L MeJA (MJ-75), 150 μmol/L MeJA (MJ-150) and 300 μmol/L MeJA (MJ-300), inoculating 2g long-tube Bulbus Lycoridis tissue culture seedling to each bottle of liquid culture medium, inoculating 3 bottles of liquid culture medium for each different treatment, setting 3 times, repeating at 25 deg.C and 50 μmol/m2The light intensity is changed into the light intensity of 110r/min for 12 hours. Culturing in liquid culture medium for 7d, 14d and 30d, taking out, and determining the content of three alkaloids including galanthamine, lycoramine and lycorine by chromatograph connected with two-stage mass spectrometer in series.
TABLE 4 content of galanthamine in Lycoris radiata tissue culture seedlings (μ g)-1DW)
TABLE 5 Likelamin content (μ g. g) in Lycoris longituba tissue culture seedlings-1DW)
TABLE 6 content of lycorine (μ g. g) in Lycoris longituba tissue culture seedlings-1DW)
As shown in FIGS. 1-3, the levels of Galantamine (GAL), Lycorine (LYC), and Lycoramine (LYCM) were highest at 7 days of MJ-75 treatment, 2.70 times, 2.01 times, and 2.85 times higher than those at MJ-0 treatment, respectively. From this, it was found that the treatment with MJ-75 for 7 days promoted the accumulation of galanthamine, lycorine and lycoramine in Lycoris radiata seedlings.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A method for improving alkaloid content in lycoris radiata by using a foreign substance is characterized in that the tissue culture seedling of 3-month-1-year-old lycoris radiata is treated by using the foreign substance, and the foreign substance is 4' -O-methyl noranthurium and/or methyl jasmonate.
2. The method according to claim 1, wherein the exogenous 4' -O-methylnoralopetridine is present in an effective treatment concentration of 0.1 to 0.2 g/L.
3. The method according to claim 1, wherein the exogenous methyl jasmonate is treated at an effective concentration of 75 to 150 μmol/L.
4. The method as claimed in claim 1, wherein the method for processing the tissue culture seedling by the exogenous material is to place the tissue culture seedling on a liquid culture medium for continuous shaking culture.
5. The method of claim 4, wherein the liquid medium is MS +6-BA 2.5-3.5 mg/L + NAA 0.8-1.5 mg/L + 25-35 g/L sucrose.
6. The method according to claim 4, wherein the shaking treatment time is 7-30 d.
7. The method as claimed in claim 6, wherein the speed of the oscillating process is 100-120 rpm/min.
8. The method as claimed in claim 1, wherein the method for culturing the tissue culture plantlet comprises inoculating the sterilized lycoris radiata seeds on a culture medium to induce protocorm, cutting the protocorm into small pieces, inoculating the small pieces of protocorm to the culture medium to induce adventitious buds, inoculating the induced adventitious buds to the culture medium to proliferate the adventitious buds, inoculating the proliferated adventitious buds to the culture medium to perform expanding culture, and obtaining the tissue culture plantlet after the expanding culture.
9. The method of claim 8, wherein the medium for performing the expansion culture is MS + 4.5-5.8 mg/L6-BA + 1-2 mg/L NAA + 0.5-2 mg/L CPPU + 25-35 g/L sucrose + 7-9 g/L agar.
10. The method according to claim 8, wherein the culture temperature of the expansion culture is 22-24 ℃, and the illumination intensity is 40-55 μmol/m2/s。
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