CN112005884B - Method for improving alkaloid content of subprostrate sophora tissue culture seedling by utilizing selenium - Google Patents
Method for improving alkaloid content of subprostrate sophora tissue culture seedling by utilizing selenium Download PDFInfo
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Classifications
-
- 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
-
- 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
- A01H4/001—Culture apparatus for tissue culture
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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a method for improving alkaloid content of subprostrate sophora tissue culture seedlings by utilizing selenium. The selenium element is added into the culture medium, so that the alkaloid content of the subprostrate sophora is effectively improved, the selenium absorption rate of plants is improved, and the growth of subprostrate sophora tissue culture seedlings, and the accumulation of in-vivo enzymes, hormones and main active ingredients can be regulated.
Description
Technical Field
The invention relates to the field of biotechnology. More specifically, the invention relates to a method for improving alkaloid content of subprostrate sophora tissue culture seedlings by utilizing selenium.
Background
Radix Sophorae Tonkinensis is famous genuine medicinal material in Guangxi province and common bulk traditional Chinese medicine, also called radix Sophorae Tonkinensis, which is leguminous plant Sophora tonkinensisSophora tonkinensisDried roots and rhizomes of gagnep. Bitter and cold in flavor. It enters lung and stomach meridians. Has effects of clearing away heat and toxic materials, relieving swelling, and relieving sore throat, and can be used for treating fire toxin accumulation, tonsillitis pharyngitis, sore throat, gingival swelling and pain, and aphtha of the mouth and tongue. Is the main raw material of nearly 30 Chinese patent medicine products for treating hepatitis, swollen sore throat, tumor, hemorrhoids, pimples, skin diseases and the like, and is used as decoction pieces in a plurality of prescriptions. The subprostrate sophora grows slowly naturally, wild resources of the subprostrate sophora are reduced year by year due to long-term felling, and the current annual output cannot meet the increasing requirements of people along with the annual increase of the market demand and price of the subprostrate sophora.
The wild subprostrate sophora has narrow wild distribution range and is sporadically distributed only in some limestone areas in Guangxi, Guizhou and Yunnan, and karst vegetation with calcareous, lithologic and xerophyte characteristics is formed in the areas. The selenium application can effectively relieve the inhibition effect of drought stress on the plant growth, the selenium and the drought stress have a synergistic effect, and the effect of relieving the drought stress is the best when the drought stress and the 4mg/kg selenium are applied together. Meanwhile, selenium can relieve the toxic action of heavy metal of plants, wherein the selenium can combine with the heavy metal into an insoluble precipitate or change the form of the heavy metal, reduce the intake of the heavy metal of plants or block the transportation process of the heavy metal, reduce the concentration of movable heavy metal ions in cells, and further achieve the technical effect of improving the growth situation of the subprostrate sophora.
Alkaloid substance, an important pharmacological component of subprostrate sophora, has various effects of resisting tumor, inflammation, virus, bacteria and the like. In the prior art, alkaloid precursors (amino acids) are introduced to improve the alkaloid content in plants, and the cells and tissue structures of the plants are diversified, so that the externally introduced alkaloid precursors (amino acids) can possibly generate synthetic reactions of other byproducts, and the plants have poor absorption rate of externally synthesized alkaloids, and finally the alkaloid content regulation effect is poor;
the addition of a certain amount of selenium can effectively improve the content of sophocarpidine and oxymatrine, but if the selenium concentration is too high, the selenium absorption and utilization rate is reduced, especially the toxicity of inorganic selenium concentration on plant cells is improved, and further the growth and development of plants are inhibited; how to achieve the dual superior effects of improving the growth and development situation of plants and simultaneously increasing the alkaloid content in the plants by adding selenium is a technical difficulty to be broken through in the field.
Disclosure of Invention
An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.
The invention also aims to provide a method for improving the alkaloid content of the subprostrate sophora tissue culture seedling by utilizing selenium, the invention effectively improves the alkaloid content of the subprostrate sophora by adding selenium element into a culture medium, simultaneously greatly improves the selenium absorption rate of plants, and can adjust the growth, in vivo enzyme and hormone, and main active ingredient accumulation of the subprostrate sophora tissue culture seedling; further, the balance of double effects of improving the growth and development situation of plants and increasing the alkaloid content in the plants is achieved by adjusting the adding amount of selenium in the culture medium, so that the effect of the selenium is maximized.
To achieve these objects and other advantages in accordance with the purpose of the invention, there is provided a method for increasing alkaloid content of tissue culture seedlings of vietnamese sophora root by using selenium, comprising culturing the tissue culture seedlings of vietnamese sophora root in a culture medium containing selenium element.
Preferably, the method for improving the alkaloid content of the subprostrate sophora root tissue culture seedling by utilizing selenium comprises the step of culturing the subprostrate sophora root tissue culture seedling in a culture medium containing sodium selenite.
Preferably, in the method for improving the alkaloid content of the subprostrate sophora tissue culture seedling by using selenium, the culture medium is 1/2MS + sodium selenite 0-40 mg/L + NAA1.0mg/L + IBA0.3mg/L + sucrose 20.0g/L + agar powder 4.0g/L and active carbon 0.5 g/L.
Preferably, the method for improving the alkaloid content of the subprostrate sophora tissue culture seedling by utilizing selenium comprises 1/2MS + sodium selenite 10-30 mg/L + NAA1.0mg/L + IBA0.3mg/L + sucrose 20.0g/L + agar powder 4.0g/L and active carbon 0.5 g/L.
Preferably, the method for improving the alkaloid content of the subprostrate sophora tissue culture seedling by utilizing selenium comprises 1/2MS, sodium selenite 20mg/L, NAA1mg/L, IBA0.3mg/L, sucrose 20g/L, agar powder 4g/L and active carbon 0.5 g/L.
Preferably, the method for improving the alkaloid content of the subprostrate sophora root tissue culture seedling by utilizing selenium comprises the steps of placing the subprostrate sophora root tissue culture seedling in the culture medium, and culturing for 45 days under the conditions that the temperature is 22-28 ℃, the illumination time is 12h/d and the illumination intensity is 2500-3000 Lux.
Preferably, the method for improving the alkaloid content of the subprostrate sophora tissue culture seedling by utilizing selenium further comprises a phomopsis elicitor in a weight ratio of 1.2: 1.
Preferably, the method for increasing the alkaloid content of the subprostrate sophora tissue culture seedling by using selenium comprises the following steps:
step one, taking 100 parts of potatoes, 10 parts of glucose, 10 parts of agar and 500 parts of water according to parts by weight, uniformly stirring to obtain a first culture medium, sequentially adding 2 parts of carbon nanotubes into the first culture medium, and fully stirring under the protection of sterile gas to obtain a second culture medium; mixing 4 parts of phomopsis sp with a second culture medium, culturing for 12d at the temperature of 25 ℃ under the dark condition, filtering to remove filtrate, washing the filter residue for 3 times by using sterile water, crushing the washed filter residue, and drying at low temperature to obtain a phomopsis elicitor-carbon nano tube compound;
step two, preparing a basic culture medium of 1/2MS + NAA1mg/L + IBA0.3mg/L + sucrose 20g/L + agar powder 4g/L + active carbon 0.5g/L, adding a phomopsis mimosa elicitor-carbon nano tube compound and sodium selenite into the basic culture medium, and culturing for 4 days on a shaking table at the rotating speed of 110r/min to obtain a tissue culture medium; the concentration of the sodium selenite is 20 mg/L; the weight ratio of the phomopsis elicitor contained in the added phomopsis elicitor-carbon nano tube compound to the sodium selenite is 1.2: 1;
and step three, inoculating the tissue culture seedling of the subprostrate sophora in a tissue culture medium, and culturing for 45d under the conditions that the temperature is 22-28 ℃, the illumination time is 12h/d and the illumination intensity is 2500-3000 Lux.
The invention at least comprises the following beneficial effects:
1. in the prior art, selenium is generally applied to soil for plant growth in the form of fertilizer, and the absorptivity is poor; according to the invention, selenium element is added into the culture medium, so that the alkaloid content of the subprostrate sophora is effectively improved, the selenium absorption rate of plants is improved, and the growth of subprostrate sophora tissue culture seedlings, and the accumulation of in-vivo enzymes, hormones and main active ingredients can be regulated; further, the balance of double effects of improving the growth and development situation of plants and increasing the alkaloid content in the plants is achieved by adjusting the adding amount of selenium in the culture medium, so that the effect of the selenium is maximized;
2. inorganic selenium sodium selenite is added into a culture medium to provide selenium element, however, the toxicity of the sodium selenite is high, and the amount of toxins deposited inside the plant is increased along with the increase of the added amount, so that the growth and development of the tissue culture seedlings are influenced; the phomopsis elicitor is introduced and is cultured with sodium selenite, the sodium selenite can be detoxified and converted by the phomopsis elicitor microbial strain in the culture process, inorganic selenium can be converted into selenium amino acid, selenium polysaccharide and selenium protein, the toxicity is greatly reduced, and the absorption rate of plants to the selenium can be improved;
3. furthermore, the phomopsis elicitor is adopted to detoxify sodium selenite and convert the sodium selenite into biological selenium, and simultaneously can interact with cells of the subprostrate sophora tissue culture seedling, can be combined with extracellular specific receptor protein, then stimulates callus to secrete signal molecules, and further regulates and controls related genes of secondary metabolic pathways, so that synthesis and accumulation of specific secondary metabolite alkaloids in the plant body are promoted, and finally the alkaloid content in the subprostrate sophora tissue culture seedling is obviously improved.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
It is to be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified.
< example 1>
The invention provides a method for improving alkaloid content of subprostrate sophora tissue culture seedlings by utilizing selenium, which comprises the following steps:
step one, preparing 1/2MS + NAA1mg/L + IBA0.3mg/L + sucrose 20g/L + agar powder 4g/L + active carbon 0.5g/L basic culture medium;
and step two, inoculating the subprostrate sophora tissue culture seedlings into a basic culture medium, and culturing for 45d under the conditions that the temperature is 22-28 ℃, the illumination time is 12h/d and the illumination intensity is 2500-3000 Lux.
< example 2>
The invention provides a method for improving alkaloid content of subprostrate sophora tissue culture seedlings by utilizing selenium, which comprises the following steps:
step one, preparing a 1/2MS + NAA1mg/L + IBA0.3mg/L + sucrose 20g/L + agar powder 4g/L + active carbon 0.5g/L basic culture medium, and adding sodium selenite into the basic culture medium, wherein the concentration of the sodium selenite is 10mg/L, so as to obtain a tissue culture medium;
and step two, inoculating the tissue culture seedling of the subprostrate sophora into a tissue culture medium, and culturing for 45 days under the conditions that the temperature is 22-28 ℃, the illumination time is 12h/d and the illumination intensity is 2500-3000 Lux.
< example 3>
The invention provides a method for improving alkaloid content of subprostrate sophora tissue culture seedlings by utilizing selenium, which comprises the following steps:
step one, preparing a 1/2MS + NAA1mg/L + IBA0.3mg/L + sucrose 20g/L + agar powder 4g/L + active carbon 0.5g/L basic culture medium, and adding sodium selenite into the basic culture medium, wherein the concentration of the sodium selenite is 20mg/L, so as to obtain a tissue culture medium;
and step two, inoculating the tissue culture seedling of the subprostrate sophora into a tissue culture medium, and culturing for 45 days under the conditions that the temperature is 22-28 ℃, the illumination time is 12h/d and the illumination intensity is 2500-3000 Lux.
< example 4>
The invention provides a method for improving alkaloid content of subprostrate sophora tissue culture seedlings by utilizing selenium, which comprises the following steps:
step one, preparing a 1/2MS + NAA1mg/L + IBA0.3mg/L + sucrose 20g/L + agar powder 4g/L + active carbon 0.5g/L basic culture medium, and adding sodium selenite into the basic culture medium, wherein the concentration of the sodium selenite is 30mg/L, so as to obtain a tissue culture medium;
and step two, inoculating the tissue culture seedling of the subprostrate sophora into a tissue culture medium, and culturing for 45 days under the conditions that the temperature is 22-28 ℃, the illumination time is 12h/d and the illumination intensity is 2500-3000 Lux.
< example 5>
The invention provides a method for improving alkaloid content of subprostrate sophora tissue culture seedlings by utilizing selenium, which comprises the following steps:
step one, preparing a 1/2MS + NAA1mg/L + IBA0.3mg/L + sucrose 20g/L + agar powder 4g/L + active carbon 0.5g/L basic culture medium, and adding sodium selenite into the basic culture medium, wherein the concentration of the sodium selenite is 40mg/L, so as to obtain a tissue culture medium;
and step two, inoculating the tissue culture seedling of the subprostrate sophora into a tissue culture medium, and culturing for 45 days under the conditions that the temperature is 22-28 ℃, the illumination time is 12h/d and the illumination intensity is 2500-3000 Lux.
< example 6>
The invention provides a method for improving alkaloid content of subprostrate sophora tissue culture seedlings by utilizing selenium, which comprises the following steps:
step one, taking 100 parts of potatoes, 10 parts of glucose, 10 parts of agar and 500 parts of water according to parts by weight, uniformly stirring to obtain a first culture medium, sequentially adding 3 parts of carbon nanotubes into the first culture medium, and fully stirring under the protection of sterile gas to obtain a second culture medium; mixing 4 parts of phomopsis sp with a second culture medium, culturing for 12d at the temperature of 25 ℃ under the dark condition, filtering to remove filtrate, washing the filter residue for 3 times by using sterile water, crushing the washed filter residue, and drying at low temperature to obtain a phomopsis elicitor-carbon nano tube compound;
step two, preparing a 1/2MS + NAA1mg/L + IBA0.3mg/L + sucrose 20g/L + agar powder 4g/L + active carbon 0.5g/L basic culture medium, adding a phomopsis mimosoides-carbon nano tube compound and sodium selenite into the basic culture medium, and culturing for 4 days on a shaking table at the rotating speed of 110r/min to obtain a tissue culture medium; the concentration of sodium selenite is 20mg/L, and the weight ratio of the phomopsis elicitor contained in the phomopsis elicitor-carbon nano tube compound to the sodium selenite is 1.2: 1;
and step three, inoculating the tissue culture seedling of the subprostrate sophora in a tissue culture medium, and culturing for 45d under the conditions that the temperature is 22-28 ℃, the illumination time is 12h/d and the illumination intensity is 2500-3000 Lux.
< example 7>
The invention provides a method for improving alkaloid content of subprostrate sophora tissue culture seedlings by utilizing selenium, which comprises the following steps:
step one, taking 100 parts of potatoes, 10 parts of glucose, 10 parts of agar and 500 parts of water according to parts by weight, uniformly stirring to obtain a first culture medium, sequentially adding 3 parts of carbon nanotubes into the first culture medium, and fully stirring under the protection of sterile gas to obtain a second culture medium; mixing 4 parts of phomopsis sp with a second culture medium, culturing for 12d at the temperature of 25 ℃ under the dark condition, filtering to remove filtrate, washing the filter residue for 3 times by using sterile water, crushing the washed filter residue, and drying at low temperature to obtain a phomopsis elicitor-carbon nano tube compound;
step two, preparing a basic culture medium of 1/2MS + NAA1mg/L + IBA0.3mg/L + sucrose 20g/L + agar powder 4g/L + active carbon 0.5g/L, adding a phomopsis mimosa elicitor-carbon nano tube compound into the culture medium, and culturing for 4 days on a shaking table at the rotating speed of 110r/min to obtain a tissue culture medium; the concentration of the phomopsis elicitor contained in the added phomopsis elicitor-carbon nano tube compound is 24 mg/L;
and step three, inoculating the tissue culture seedling of the subprostrate sophora in a tissue culture medium, and culturing for 45d under the conditions that the temperature is 22-28 ℃, the illumination time is 12h/d and the illumination intensity is 2500-3000 Lux.
< example 8>
The invention provides a method for improving alkaloid content of subprostrate sophora tissue culture seedlings by utilizing selenium, which comprises the following steps:
step one, taking 100 parts of potatoes, 10 parts of glucose, 10 parts of agar and 500 parts of water according to parts by weight, and uniformly stirring to obtain a first culture medium; mixing 4 parts of phomopsis longissima strain with a first culture medium, culturing for 12d at the temperature of 25 ℃ in the dark, filtering to remove filtrate, washing filter residue for 3 times by using sterile water, crushing the washed filter residue, and drying at low temperature to obtain phomopsis longissima elicitor;
step two, preparing a 1/2MS + NAA1mg/L + IBA0.3mg/L + sucrose 20g/L + agar powder 4g/L + active carbon 0.5g/L basic culture medium, adding a phomopsis mimosoides elicitor and sodium selenite into the basic culture medium, and culturing for 4 days on a shaking table at the rotating speed of 110r/min to obtain a tissue culture medium; the concentration of sodium selenite is 20mg/L, and the weight ratio of the added phomopsis elicitor to the sodium selenite is 1.2: 1;
and step three, inoculating the tissue culture seedling of the subprostrate sophora in a tissue culture medium, and culturing for 45d under the conditions that the temperature is 22-28 ℃, the illumination time is 12h/d and the illumination intensity is 2500-3000 Lux.
< test example one >
Carrying out 5 groups of subprostrate sophora root tissue culture seedling culture experiments by using the culture methods of examples 1-5 respectively, and numbering T0, T1, T2, T3 and T4; for each group of experiments, 30 bottles of tissue culture medium are arranged in parallel, 6 subprostrate sophora tissue culture seedlings are inoculated in each bottle, and the quality of the subprostrate sophora tissue culture seedlings inoculated in all the experimental bottles is consistent;
1. the growth characteristics (average root length, average root number, rooting rate, survival rate) of the subprostrate sophora obtained in 5 experiments (T0-T4) were analyzed and counted, and the results are shown in Table 1:
TABLE 1 growth traits of Sophora Subprostrata
Remarking: average root length (mm/plant) = total root length/total number of plants;
average number of roots (root/plant) = total number of roots/total number of plants;
rooting rate (%) = total rooted plant number/total plant number × 100%;
survival (%) = total viable plant number/total plant number × 100%.
As can be seen from the data in Table 1, the change of selenium concentration has a great influence on the rooting condition of the tissue culture seedling of subprostrate sophora, and among 5 subprostrate sophora tissue culture seedlings cultured at different selenium concentrations, T1, example 2 (concentration of 10 mg/L) has the best average root length of subprostrate sophora, T2, example 3 has the lowest average root length (concentration of 20 mg/L), and T0, example 1 (concentration of 0 mg/L) has the shortest average root length; t3- -the rooting percentage of Sophora subprostrata was the best in example 4 (concentration of 30 mg/L), the second best in T1- -example 2 (concentration of 10 mg/L) and the lowest in T0- -example 1 (concentration of 0 mg/L). Therefore, when selenium (10 mg/L) with proper concentration is added into the culture medium, the subprostrate sophora root tissue culture seedling has the best rooting and survival conditions, the rooting and survival conditions are obviously different from those of the subprostrate sophora root tissue culture seedling without selenium, the average root length, the average root number and the rooting rate of the subprostrate sophora root tissue culture seedling are low under the treatment of the selenium (40 mg/L) with high concentration, and the root is black.
2. Analyzing and counting the physiological and biochemical indexes of the subprostrate sophora obtained in 5 groups of experiments (T0-T4), and the results are shown in Table 2;
TABLE 2 physiological and biochemical indexes of radix Sophorae Tonkinensis
Remarking: POD-peroxidase; SOD-superoxide dismutase; GOT-glutamic oxaloacetic transaminase; GPT-glutamate pyruvate transaminase; MDA-malondialdehyde; ABA-abscisic acid; GA-gibberellin; ZA-zeatin; BR-brassinolide; JA-ME-methyl jasmonate.
As can be seen from Table 2:
and (3) POD: selenium is not only positively or negatively correlated with Peroxidase (POD) activity of the subprostrate sophora tissue culture seedling, and the change trend of the peroxidase activity is firstly increased and then decreased within the selenium concentration range of 10-40 mg/L, T1 has the highest POD activity (29660.1527U/gFW/min) corresponding to example 2 (sodium selenite concentration is 10 mg/L), T4 has the lowest POD activity (28653.9849U/gFW/min) corresponding to example 5 (sodium selenite concentration is 40 mg/L), and has obvious POD activity inhibition effect compared with T0 (example 1 (no sodium selenite is added).
SOD (superoxide dismutase): selenium has obvious enhancement effect on superoxide dismutase (SOD) activity of the subprostrate sophora tissue culture seedling within a certain range, T1 is the highest SOD activity (452.9876U/gFW/min) corresponding to example 2 (the concentration of sodium selenite is 10 mg/L), T2 is the lowest SOD activity (450.4831U/gFW/min) corresponding to example 3 (the concentration of sodium selenite is 20 mg/L), and T0 is the lowest SOD activity corresponding to example 1 (no sodium selenite is added). The significance of the homogeneity test of the variance is 0.095, and the results satisfy the original hypothesis, and the variance test shows that the SOD activities corresponding to T1, T2, T3 and T4 (examples 2 to 5) are significantly different from the SOD activities corresponding to T0-example 1 (sodium selenite is not added) (P < 0.05).
GOT: the activity of glutamic-oxaloacetic transaminase (GOT) in the subprostrate sophora tissue culture seedlings tends to decrease first and then increase along with the increase of the selenium concentration. The GOT activity of the subprostrate sophora reaches the maximum (2.9868 mg/gFW/30 min) when the selenium concentration is increased from 0mg/L to 10mg/L, and the GOT activity starts to decrease when the selenium concentration is increased continuously. The significance of the homogeneity test of variance is 0.609, and the original hypothesis is satisfied, and the variance test shows that the GOT activity (P < 0.05) of T1, example 2, T3, example 4 and T4, example 5, is different from that of T0, example 1.
GPT: similar to the variation trend of GOT activity, the activity of subprostrate sophora root Glutamic Pyruvic Transaminase (GPT) is increased to 1.4043mg/gFW/30min when the concentration of sodium selenite is 10mg/L, is greatly reduced to 0.2145mg/gFW/30min when the concentration of sodium selenite is 20mg/L, the selenium concentration is continuously increased, the activity of the GPT is recovered and increased, and the activity of the GPT reaches the maximum value (1.7016 mg/gFW/30 min) when the concentration of the selenium is 40 mg/L. According to the data, the GPT activity of the subprostrate sophora root tissue culture seedling is the best when the selenium concentration is 40mg/L, and the GPT activity of the subprostrate sophora root tissue culture seedling treated by 10mg/L selenium is the lowest when the selenium concentration is 20 mg/L. Analysis of variance results indicated that the selenium concentration of the T1, example 2, T3, example 4, T4, example 5 treatments was significantly different from the GPT activity of the T0 treatment (P < 0.05).
MDA: the content of Malondialdehyde (MDA) can be used for measuring the peroxidation degree of cell membrane lipid and the strong and weak response to adverse conditions. When the selenium concentration is 30mg/L, the MDA content of the subprostrate sophora tissue culture seedling is the highest (0.0031 mmol/g), the selenium concentration is continuously increased to 40mg/L, the MDA content is remarkably reduced to the lowest (0.0026 mmol/g), the significance of the variance homogeneity test is 0.374, the original hypothesis is met, and the variance test shows that the MDA content has no significant difference compared with the T0-example 1 treatment (P > 0.05) under each concentration treatment.
Soluble protein: the percentage of soluble protein varied significantly with increasing selenium concentration, and did not simply increase or decrease, with the percentage reaching a maximum at a selenium concentration of 10mg/L (2.5853%) and a minimum without sodium selenite (1.8682%). Anova showed a significant difference in soluble protein content with different concentrations of selenium compared to that without sodium selenite (P < 0.05).
Soluble sugar: the change trend of the soluble sugar is different, the selenium concentration is gradually reduced within the treatment range of 0-30 mg/L, the selenium concentration reaches the lowest value (2.2722%) when the concentration is 30mg/L, and the selenium concentration is the highest value (3.1893%) when the concentration is 20 mg/L. The significance of the homogeneity test of variance is 0.142, and the original hypothesis is satisfied, and the variance test shows that the soluble sugar content of T1, example 2, T3, example 4 and T4, example 5 is significantly different from that of T0, example 1 (P < 0.05).
Chlorophyll: the chlorophyll content of the tissue culture seedling of the subprostrate sophora treated by different selenium concentrations is not in simple positive correlation or negative correlation, the selenium concentration is in the treatment range of 0-20 mg/L, the content of the tissue culture seedling of the subprostrate sophora is in the trend of increasing firstly and then decreasing, and the tissue culture seedling of the subprostrate sophora treated by 10mg/L selenium is the highest (0.3290 mg/g); the concentration is increased continuously, the chlorophyll content is increased firstly and then decreased, and the maximum value (0.3647 mg/g) is reached when the selenium is treated at 30 mg/L. Variance tests were performed to show that the chlorophyll content of the selenium treatments at different concentrations was significantly different from that of the T0 treatment of example 1 (P < 0.05).
Endogenous hormone abscisic acid (ABA): the selenium concentration is within the treatment range of 0-40 mg/L, the change trend of abscisic acid (ABA) of the subprostrate sophora tissue culture seedlings is increased firstly and then decreased, the ABA content is the lowest value (6.4321 mu g/g) during 10mg/L selenium treatment, the concentration is continuously increased, and the ABA value is continuously increased. Analysis of variance tests showed significant differences in the concentration of selenium in T1, examples 2 and T4, example 5 versus T0, the ABA content of vietnamese sophora root treated in example 1 (P < 0.05).
GA: the content of Gibberellin (GA) in the tissue culture seedling of subprostrate sophora is decreased and then increased along with the increase of the selenium concentration, and the content is the lowest value (19.2181 ng/gFW) in the case of selenium treatment at 20mg/L and the highest value (31.3673 ng/gFW) in the case of selenium treatment at 40 mg/L. Analysis of variance tests showed significant differences in GA content only for T4, example 5 versus T0, example 1 (P < 0.05), and no significant differences for T1, example 2, T2, example 3, T3, example 4.
ZA: the selenium concentration is within the treatment range of 10-40 mg/L, the content of the subprostrate sophora root tissue culture seedling Zeatin (ZA) is increased and then reduced along with the change of the selenium concentration, the content of the subprostrate sophora root ZA reaches the maximum value (6.3872ng/gFW) when the selenium concentration is 30mg/L, and the content of the subprostrate sophora root ZA is the minimum value (5.1023 ng/gFW) when the selenium concentration is 20 mg/L. The significance of the homogeneity test of the variance is 0.998, the requirement is met, but the difference of the average values shows that the difference of the different selenium concentrations on the change of the content of the subprostrate sophora root tissue culture seedling ZA is not significant.
BR: along with the increase of the selenium concentration, the content of Brassinolide (BR) of the tissue culture seedling of the subprostrate sophora is firstly reduced and then increased, the lowest value (1.0844 ng/gFW) is obtained when 20mg/L selenium treatment is carried out, the highest value (1.3878 ng/gFW) is obtained when 0mg/L selenium treatment is carried out, and the highest value (1.1691 ng/gFW) is obtained when 10mg/L selenium treatment is carried out. Variance tests were performed to show that the BR content was significantly different for the selenium treatments at different concentrations compared to the BR content of T0, example 1 treatment (P < 0.05).
JA-ME: along with the increase of the selenium concentration, the content of the methyl jasmonate (JA-ME) of the tissue culture seedling of the subprostrate sophora is increased firstly and then reduced, the highest value is obtained when 10mg/L of selenium is treated (8.5436 pmol/gFW), and the lowest content is obtained when 40mg/L of selenium is treated. The significance of the variance test showed that each group of treatments differed significantly from the JA-ME content of T0, example 1 treatment (P > 0.05).
3. The alkaloid content of the subprostrate sophora root obtained by 5 groups of experiments (T0, T1, T2 and T4) is analyzed and counted, and the result is shown in Table 3;
TABLE 3 Sophora Subprostrata root alkaloid content
From the data in table 3, it can be seen that:
from the whole plant measurement results, the application of selenium element has obvious influence on the content accumulation of both Matrine (MA) and Oxymatrine (OMA) of the subprostrate sophora tissue culture seedling, the content of both the MA and the M of the subprostrate sophora shows a trend of increasing first and then decreasing with the increase of the selenium concentration, the maximum value is reached when the subprostrate sophora tissue culture seedling is treated by 20mg/L selenium, and the content of main MA and OMA in the subprostrate tissue culture seedling added with the selenium element is obviously higher than that treated by T0, which shows that the 20mg/L selenium element is the optimal concentration for promoting the synthesis and accumulation of the matrine of the subprostrate tissue culture seedling.
The determination result of the stem and leaf part shows that the content of OMA and M increases and then decreases when the selenium concentration is increased, the OMA content of the subprostrate sophora reaches the maximum value when the selenium is treated at 10mg/L, the MA and M content of the subprostrate sophora reaches the maximum value when the selenium is treated at 20mg/L, and the optimal accumulation concentration of the selenium element for synthesizing and accumulating the secondary metabolites MA and OMA at the stem and leaf part is 20mg/L through comprehensive analysis.
From the root measurement results, it is known that the contents of MA, OMA and M in the roots of the tissue culture seedlings of Sophora subprostrata all increased with the increase of the selenium concentration, and the contents of MA, OMA and M in the roots of the Sophora subprostrata all reached the maximum value when the selenium is treated at 40 mg/L.
Comparing the whole data of the whole plant, the stem leaves and the roots, the MA and the OMA are mainly enriched in the stem leaves and have lower root content; the method takes M as a main index, integrates physiological and biochemical indexes and agronomic indexes of each group of subprostrate sophora tissue culture seedlings, adds effective components which are most suitable for the growth and accumulation of the subprostrate sophora tissue culture seedlings and can be used as an important regulating factor for producing secondary metabolites on a large scale by the subprostrate sophora tissue culture seedlings by adding 20mg/L selenium.
< second test example >
Carrying out culture experiments on 4 groups of subprostrate sophora tissue culture seedlings by using the culture methods of the embodiments 3 and 6-8 respectively; for each group of experiments, 30 bottles of tissue culture medium are arranged in parallel, 6 subprostrate sophora tissue culture seedlings are inoculated in each bottle, and the quality of the subprostrate sophora tissue culture seedlings inoculated in all the experimental bottles is consistent;
analyzing and counting the average root length, the average root number, the rooting rate and the alkaloid content of the whole plant of the subprostrate sophora cultured by the B1-B4, and recording the results to be shown in a table 4;
TABLE 4 statistical results
| Index (I) | Example 3 | Example 6 | Example 7 | Example 8 |
| Average root length (mm/strain) | 3.56 | 3.74 | 0.63 | 3.61 |
| Average root number (root/plant) | 2.43 | 4.37 | 1.21 | 3.97 |
| Rooting percentage (%) | 63.29 | 85.61 | 11.92 | 83.21 |
| Matrine (MA) mg/g | 6.1370±0.0757 | 7.2813±0.0365 | 4.3515±0.0539 | 6.5132±0.0378 |
| Oxymatrine (OMA) mg/g | 13.6475±0.3243 | 15.6721±0.1437 | 11.1867±0.1069 | 14.3791±0.1352 |
| M（MA + OMA）mg/g | 19.7845±0.3872 | 21.7522±0.2391 | 14.2082±0.2156 | 20.2985±0.2219 |
Remarking: average root length (mm/plant) = total root length/total number of plants;
average number of roots (root/plant) = total number of roots/total number of plants;
rooting percentage (%) = total rooted plants/total plants × 100%.
As can be seen from the data in table 4, the subprostrate sophora obtained by the cultivation method provided in example 6 has more excellent effects on improving growth characteristics and pharmacological component alkaloid accumulation, and compared with the indexes corresponding to example 1 (see comparative table 1 and table 3) and example 7, the alkaloid content of the subprostrate sophora obtained in example 7 is significantly increased, which indicates that the alkaloid content of the subprostrate sophora can be increased by adding the phomopsis inducer into the culture medium; comparing example 6, example 8 and example 3, it is known that the introduction of sodium selenite detoxified by phomopsis elicitor cultured on carbon nanotubes into the tissue culture medium can improve the growth characteristics of vietnamese sophora root and increase the alkaloid content of vietnamese sophora root.
The number of apparatuses and the scale of the process described herein are intended to simplify the description of the present invention. Applications, modifications and variations of the present invention will be apparent to those skilled in the art.
While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.
Claims (1)
1. The method for improving the alkaloid content of the subprostrate sophora tissue culture seedling by utilizing selenium is characterized by comprising the following steps of:
step one, taking 100 parts of potatoes, 10 parts of glucose, 10 parts of agar and 500 parts of water according to parts by weight, uniformly stirring to obtain a first culture medium, sequentially adding 2 parts of carbon nanotubes into the first culture medium, and fully stirring under the protection of sterile gas to obtain a second culture medium; mixing 4 parts of phomopsis sp with a second culture medium, culturing for 12d at the temperature of 25 ℃ under the dark condition, filtering to remove filtrate, washing the filter residue for 3 times by using sterile water, crushing the washed filter residue, and drying at low temperature to obtain a phomopsis elicitor-carbon nano tube compound;
step two, preparing a basic culture medium of 1/2MS + NAA1mg/L + IBA0.3mg/L + sucrose 20g/L + agar powder 4g/L + active carbon 0.5g/L, adding a phomopsis mimosa elicitor-carbon nano tube compound and sodium selenite into the basic culture medium, and culturing for 4 days on a shaking table at the rotating speed of 110r/min to obtain a tissue culture medium; the concentration of the sodium selenite is 20 mg/L; the weight ratio of the phomopsis elicitor contained in the added phomopsis elicitor-carbon nano tube compound to the sodium selenite is 1.2: 1;
and step three, inoculating the tissue culture seedling of the subprostrate sophora in a tissue culture medium, and culturing for 45d under the conditions that the temperature is 22-28 ℃, the illumination time is 12h/d and the illumination intensity is 2500-3000 Lux.
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