CN113498736B - Method for inducing and culturing adventitious roots of astragalus membranaceus - Google Patents
Method for inducing and culturing adventitious roots of astragalus membranaceus Download PDFInfo
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Abstract
The invention discloses an inducing and culturing method of astragalus adventitious roots, which comprises the following steps: 1) Culturing the astragalus aseptic seedlings; inoculating the sterilized astragalus seeds on an aseptic seedling culture medium, and culturing to obtain astragalus aseptic seedlings; 2) Induction of callus: cutting off hypocotyl of aseptic seedling of radix astragali, inoculating to callus induction culture medium, culturing, and growing callus; 3) Induction of adventitious roots: transferring the callus to an adventitious root induction culture medium, culturing and growing adventitious roots; 4) Culturing adventitious roots: cutting and inoculating the adventitious roots into a growth culture medium for the first adventitious root culture, and then transferring the adventitious roots into a new growth culture medium for continuous culture and harvesting; the method for obtaining the astragalus adventitious roots has the characteristics of simple process, high inductivity, quick proliferation of the adventitious roots, high yield, short production period and low energy consumption, can realize large-scale production, and effectively solves the contradiction between the requirement of the astragalus on raw materials for industrially extracting effective components and wild resource protection.
Description
Technical Field
The invention discloses an induction and culture method of astragalus adventitious roots, belonging to the field of tissue culture of astragalus membranaceus, a traditional Chinese medicine.
Background
Astragalus membranaceus is a perennial herb of genus Astragalus of family Leguminosae, and is used as a main root. The astragalus root is the top grade of traditional Chinese medicine, has long medicinal history and is widely applied clinically. Modern pharmacological studies show that astragalus has the effects of tonifying qi, consolidating exterior, promoting urination, detoxifying, healing sore, promoting granulation, tonifying qi and strengthening middle-jiao, and can promote humoral immunity and cellular immunity of organisms. At present, with increasingly successful sale of health care medicines, the dosage of astragalus membranaceus is larger and larger, natural resources are scarce, 4-5 years are generally needed from sowing to harvesting in the seed propagation of astragalus membranaceus, the emergence rate is very low (30% -40%), and the influence of plant diseases and insect pests is serious, so that the quality and the yield are reduced. The active ingredients of the astragalus membranaceus produced by using a tissue culture method have great development prospect, and can meet the requirements of domestic and foreign markets. At present, researches such as transplanting, cuttage, callus induction and the like are carried out, but the method has a longer plant growth period or lower active ingredient content, and at present, a complete and detailed astragalus adventitious root induction and culture method is not reported.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provide a rapid, feasible and uniform culture, can be industrially produced, provide an inducing and culturing method for the adventitious roots of astragalus with high content of astragaloside and the like, and provide sufficient raw materials for extracting active ingredients of astragalus.
The technical scheme of the invention is summarized as follows:
an induction and culture method of astragalus adventitious roots comprises the following steps:
1) Culturing the astragalus aseptic seedlings;
inoculating sterilized radix astragali seed to sterile seedling culture medium, culturing at 20-30 deg.C under illumination of 1000-2000Lux for 12-16 hr in darkness for 10-20 days to obtain radix astragali sterile seedling;
2) Induction of callus: cutting off hypocotyl of aseptic seedling of radix astragali, inoculating to callus induction culture medium, and culturing at 20-30 deg.C in dark to obtain callus;
3) Induction of adventitious roots: transferring the callus obtained in the step 2) into an adventitious root induction culture medium, and carrying out dark culture at 20-30 ℃ for 30-40 days to grow adventitious roots;
4) Culturing adventitious roots: cutting and inoculating the adventitious roots obtained in the step 3) into a growth culture medium, wherein the first adventitious root culture is carried out after 30-40 days of growth, and then, transferring the adventitious roots obtained by the first adventitious root culture into a new growth culture medium for continuous culture and harvesting;
the sterile seedling culture medium is prepared by the following method:
adding 6-benzylaminopurine into MS culture medium to make final concentration be 1.0-2.0mg/L, adding indolebutyric acid to make final concentration be 0.15-0.3mg/L, adding sucrose to make final concentration be 20-30g/L, adding agar to make final concentration be 6-10g/L; adjusting the pH value to 5.8-6.0;
the callus induction culture medium is prepared by the following method:
adding 2,4-dichlorophenoxyacetic acid into MS culture medium to make final concentration 0.1-2.0mg/L, adding 6-benzylaminopurine to make final concentration 0.1-1.0mg/L, adding sucrose to make final concentration 20-30g/L, adding agar to make final concentration 6-10g/L; adjusting the pH value to 5.8-6.0;
the adventitious root induction culture medium is prepared by the following method:
adding indolebutyric acid into MS culture medium to make final concentration be 0.5-2.0mg/L, adding sucrose to make final concentration be 20-30g/L, adding agar to make final concentration be 6-10g/L; adjusting the pH value to 5.8-6.0;
the growth medium is prepared by the following method:
adding indolebutyric acid into MS culture medium to make final concentration be 2.0-6.0mg/L, adding kinetin to make final concentration be 0.2-0.6mg/L, adding sucrose to make final concentration be 20-30g/L; adjusting the pH value to 5.5-5.8.
The invention has the advantages that:
the method for obtaining the astragalus adventitious roots has the characteristics of simple process, high inductivity, quick proliferation of the adventitious roots, high yield, short production period and low energy consumption, can realize large-scale production, and effectively solves the contradiction between the requirement of the astragalus on raw materials for industrially extracting effective components and wild resource protection.
Detailed Description
The present invention will be further illustrated by the following specific examples.
The astragalus seeds used in each example were seeds of astragalus mongholicus purchased from astragalus mongholicus production bases of inner mongolian, but the present invention is not limited thereto.
Example 1
The sterile seedling culture medium is prepared by the following method:
adding 6-benzylaminopurine to a MS culture medium to make the final concentration 1.0mg/L, adding indolebutyric acid to make the final concentration 0.15mg/L, adding sucrose to make the final concentration 20g/L, and adding agar to make the final concentration 6g/L; adjusting the pH value to 5.8;
example 2
The sterile seedling culture medium is prepared by the following method:
adding 6-benzylaminopurine to MS culture medium to make final concentration 2.0mg/L, adding indolebutyric acid to make final concentration 0.3mg/L, adding sucrose to make final concentration 30g/L, and adding agar to make final concentration 10g/L; adjusting the pH value to 6.0;
example 3
The sterile seedling culture medium is prepared by the following method:
adding 6-benzylaminopurine to MS culture medium to make final concentration 1.5mg/L, adding indolebutyric acid to make final concentration 0.2mg/L, adding sucrose to make final concentration 30g/L, and adding agar to make final concentration 7g/L; adjusting the pH value to 6.0;
example 4
The callus induction culture medium is prepared by the following method:
adding 2,4-dichlorophenoxyacetic acid into MS culture medium to make final concentration 0.5mg/L, adding 6-benzylaminopurine to make final concentration 0.1mg/L, adding sucrose to make final concentration 20g/L, and adding agar to make final concentration 6g/L; adjusting the pH value to 5.8;
example 5
The callus induction culture medium is prepared by the following method:
adding 2,4-dichlorophenoxyacetic acid into MS culture medium to make final concentration 2.0mg/L, adding 6-benzylaminopurine to make final concentration 1.0mg/L, adding sucrose to make final concentration 30g/L, and adding agar to make final concentration 8g/L; adjusting the pH value to 6.0;
example 6
The callus induction culture medium is prepared by the following method:
adding 2,4-dichlorophenoxyacetic acid into MS culture medium to make final concentration 1.0mg/L, adding 6-benzylaminopurine to make final concentration 0.5mg/L, adding sucrose to make final concentration 30g/L, and adding agar to make final concentration 10g/L; adjusting the pH value to 6.0;
example 7
The adventitious root induction culture medium is prepared by the following method:
adding indolebutyric acid into an MS culture medium to enable the final concentration to be 0.5mg/L, adding sucrose to enable the final concentration to be 20g/L, and adding agar to enable the final concentration to be 6g/L; adjusting the pH value to 5.8;
example 8
The adventitious root induction culture medium is prepared by the following method:
adding indolebutyric acid into an MS culture medium to enable the final concentration to be 2.0mg/L, adding sucrose to enable the final concentration to be 30g/L, and adding agar to enable the final concentration to be 8g/L; adjusting the pH value to 6.0;
example 9
The adventitious root induction culture medium is prepared by the following method:
adding indolebutyric acid into an MS culture medium to enable the final concentration to be 1.0mg/L, adding sucrose to enable the final concentration to be 30g/L, and adding agar to enable the final concentration to be 10g/L; adjusting the pH value to 6.0;
example 10
The growth medium was prepared by the following method:
adding indolebutyric acid into an MS culture medium to enable the final concentration to be 2.0mg/L, adding kinetin to enable the final concentration to be 0.2mg/L, and adding sucrose to enable the final concentration to be 20g/L; the pH was adjusted to 5.5.
Example 11
The growth medium was prepared by the following method:
adding indolebutyric acid into an MS culture medium to enable the final concentration to be 6.0mg/L, adding kinetin to enable the final concentration to be 0.6mg/L, and adding sucrose to enable the final concentration to be 30g/L; the pH was adjusted to 5.8.
Example 12
The growth medium was prepared by the following method:
adding indolebutyric acid into an MS culture medium to enable the final concentration to be 4.0mg/L, adding kinetin to enable the final concentration to be 0.5mg/L, and adding sucrose to enable the final concentration to be 30g/L; the pH was adjusted to 5.8.
Example 13
An induction and culture method of astragalus adventitious roots comprises the following steps:
1) Culturing the astragalus aseptic seedlings;
and (3) sterilizing the astragalus seeds: washing radix astragali seed with tap water, soaking in 30 deg.C warm water for 4h, washing with 75% ethanol water solution for 20s and 4% sodium hypochlorite water solution for 5min for sterilization, and washing with sterile water for 3 times;
inoculating sterilized radix astragali seed to sterile seedling culture medium (prepared in example 1), and culturing at 20 deg.C under illumination intensity of 1000Lux for 16 hr and dark intensity of 8 hr for 20 days to obtain radix astragali sterile seedling;
2) Induction of callus: cutting off hypocotyl of aseptic seedling of radix astragali, inoculating on callus induction culture medium (prepared in example 4), and culturing at 20 deg.C in dark to grow callus;
3) Induction of adventitious roots: transferring the callus obtained in the step 2) to an adventitious root induction culture medium (prepared in example 7), and culturing in dark at 20 ℃ (promoting adventitious root proliferation) for about 40 days to grow adventitious roots;
4) Culturing adventitious roots: cutting the adventitious roots obtained in the step (3) into small sections of 0.5cm, inoculating the small sections into a growth culture medium (prepared in example 10) according to the mass-volume ratio of 0.7% (g/mL), culturing for 40 days as a first adventitious root, transferring the adventitious roots obtained by the first adventitious root culture into a new growth culture medium (prepared in example 10) according to the mass-volume ratio of 0.7% (g/mL), continuously culturing for 40 days, and harvesting;
the biomass of the adventitious roots of the astragalus is improved by 13.8 times compared with the inoculation amount. Drying at 60 ℃ to constant weight, storing in a plastic package bag, and taking a proper amount for content determination.
Example 14
An induction and culture method of astragalus adventitious roots comprises the following steps:
1) Culturing the astragalus aseptic seedlings;
sterilizing astragalus seeds: sterilizing the astragalus seeds of example 13;
inoculating sterilized radix astragali seed to sterile seedling culture medium (prepared in example 2), illuminating at 30 deg.C for 12 hr per day at 1500Lux for 12 hr in darkness, and culturing for 20 days to obtain radix astragali sterile seedling;
2) Induction of callus: cutting off hypocotyl of sterile seedling of radix astragali, inoculating to callus induction culture medium (prepared in example 5), and culturing at 25 deg.C in dark to obtain callus;
3) Induction of adventitious roots: transferring the callus obtained in the step 2) to an adventitious root induction culture medium (prepared in example 8), and culturing in the dark at 30 ℃ (promoting adventitious root proliferation) for about 35 days to grow adventitious roots;
4) Culturing adventitious roots: cutting the adventitious roots obtained in the step (3) into small segments of 1.0cm, inoculating the small segments into a growth medium (prepared in example 11), inoculating the small segments according to the mass-to-volume ratio of 1.0 percent ((g/mL)), growing for 30 days to obtain a first adventitious root culture, transferring the adventitious roots obtained by the first adventitious root culture according to the mass-to-volume ratio of 1.0 percent ((g/mL)) into a new growth medium (prepared in example 11), continuing to culture for 40 days, and harvesting;
the biomass of the adventitious roots of the astragalus is improved by 15 times compared with the inoculation amount. Drying at 60 ℃ to constant weight, storing in a plastic package bag, and taking a proper amount for content determination.
Example 15
An induction and culture method of astragalus adventitious roots comprises the following steps:
1) Culturing the astragalus aseptic seedlings;
and (3) sterilizing the astragalus seeds: sterilizing the astragalus seeds of example 13;
inoculating sterilized radix astragali seed to sterile seedling culture medium (prepared in example 3), culturing at 25 deg.C under illumination for 14 hr, illumination intensity of 2000Lux, and darkness for 10 hr for 10 days to obtain radix astragali sterile seedling;
2) Induction of callus: cutting off hypocotyl of aseptic seedling of radix astragali, inoculating to callus induction culture medium (prepared in example 6), and culturing at 30 deg.C in dark to obtain callus;
3) Induction of adventitious roots: transferring the callus obtained in the step 2) to an adventitious root induction culture medium (prepared in example 9), and culturing in the dark at 20 ℃ (promoting adventitious root proliferation) for about 30 days to grow adventitious roots;
4) Culturing adventitious roots: cutting the adventitious roots obtained in the step (3) into small sections of 1.0cm, inoculating the small sections into a growth culture medium (prepared in example 12) according to the mass-volume ratio of 1.5% (g/mL), culturing the first adventitious roots after 35 days of growth, transferring the adventitious roots obtained by the first adventitious roots culture into a new growth culture medium (prepared in example 12) according to the mass-volume ratio of 1.5% (g/mL), continuously culturing for 40 days, and harvesting;
the biomass of the adventitious roots of the astragalus is improved by 20 times compared with the inoculation amount. Drying at 60 ℃ to constant weight, storing in a plastic package bag, and taking a proper amount for content determination.
The extraction and content determination of astragaloside IV and calycosin glucoside in the obtained adventitious root of astragalus root are carried out according to 2020 edition Chinese pharmacopoeia:
astragaloside IV
(1) Chromatographic conditions
The chromatographic column is Kromasil C 18 (4.6 mm. Times.250mm, 5 μm); acetonitrile-water (32; detection by an evaporative light scattering detector.
(2) Preparation of Standard solutions
Accurately weighing appropriate amount of astragaloside IV reference substance, and adding 80% methanol to obtain solution containing 0.5mg per 1 mL. The reference stock solution was filtered through a 0.22 μm microporous membrane and sealed, and stored in a refrigerator for further use.
(3) Preparation of sample solution
Taking the dried adventitious roots of the astragalus membranaceus obtained in examples 13, 14 and 15, respectively crushing, sieving by a four-mesh sieve, respectively taking 1g, precisely weighing, placing in a conical flask with a plug, precisely adding 50mL of 80% methanol solution containing 4% concentrated ammonia test solution (taking 4mL of concentrated ammonia test solution, adding 80% methanol to 100mL, shaking up), sealing, weighing, heating and refluxing for 1 hour, cooling, weighing again, complementing the weight loss by 80% methanol solution containing 4% concentrated ammonia test solution, shaking up, filtering, precisely weighing 25mL of subsequent filtrate, evaporating to dryness, dissolving residue by 80% methanol, transferring to a 5mL measuring flask, adding 80% methanol to scale, shaking up, filtering, and taking subsequent filtrate. The sample solution is filtered by a 0.22 mu m microporous membrane and sealed, and then is stored in a refrigerator for later use.
The experimental results are as follows: the astragaloside IV contents in the products obtained in examples 13-15 were 0.08 + -0.002%, 0.15 + -0.005%, 0.14 + -0.010%, respectively. Can meet the requirement of 2020 edition Chinese pharmacopoeia on the content of astragaloside in astragalus (not less than 0.080%).
Calycosin glucosides
(1) Chromatographic conditions
The chromatographic column is Kromasil C 18 (4.6 mm. Times.250mm, 5 μm); acetonitrile is taken as a mobile phase A, 0.2% formic acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the detection wavelength was 260nm.
(2) Preparation of Standard solutions
Taking a proper amount of calycosin glucoside reference substance, precisely weighing, and adding methanol to obtain a solution containing 50 μ g per 1 mL. The reference stock solution was filtered through a 0.22 μm microporous membrane and sealed, and stored in a refrigerator for further use.
(3) Preparation of sample solution
Respectively pulverizing the dried adventitious roots of radix astragali obtained in examples 13, 14 and 15, sieving with a four-mesh sieve, respectively taking 1g, precisely weighing, placing in a round bottom flask, precisely adding 50mL of methanol, weighing, heating under reflux for 4 hours, cooling, weighing again, supplementing the lost weight with methanol, shaking up, filtering, precisely weighing 25mL of the subsequent filtrate, recovering solvent to dryness, dissolving the residue with methanol, transferring to a 5mL measuring flask, adding methanol to scale, and shaking up to obtain the final product. The sample solution was filtered through a 0.22 μm microporous membrane and sealed, and stored in a refrigerator for further use.
The experimental results are as follows: the products obtained in examples 13 to 15 had calycosin glucoside contents of 0.11. + -. 0.001%, 0.06. + -. 0.002%, 0.06. + -. 0.001%, respectively. Can meet the requirement of 2020 edition Chinese pharmacopoeia on the content of calycosin glucoside in astragalus (not less than 0.020%).
Claims (1)
1. An induction and culture method of astragalus adventitious roots is characterized by comprising the following steps:
1) Culturing the astragalus aseptic seedlings;
inoculating sterilized radix astragali seed to sterile seedling culture medium, culturing at 20-30 deg.C under illumination for 12-16 hr per day with illumination intensity of 1000-2000Lux and 8-12 hr in dark for 10-20 days to obtain radix astragali sterile seedling;
2) Induction of callus: cutting off hypocotyl of aseptic seedling of radix astragali, inoculating to callus induction culture medium, and culturing at 20-30 deg.C in dark to obtain callus;
3) Induction of adventitious roots: transferring the callus obtained in the step 2) into an adventitious root induction culture medium, and carrying out dark culture at 20-30 ℃ to grow adventitious roots;
4) Culturing adventitious roots: cutting and inoculating the adventitious roots obtained in the step 3) into a growth culture medium, wherein the first adventitious root culture is carried out after 30-40 days of growth, and then, transferring the adventitious roots obtained by the first adventitious root culture into a new growth culture medium for continuous culture and harvesting;
the sterile seedling culture medium is prepared by the following method:
adding 6-benzylaminopurine into MS culture medium to make final concentration be 1.0-2.0-mg/L, adding indolebutyric acid to make final concentration be 0.15-0.3mg/L, adding sucrose to make final concentration be 20-30g/L, adding agar to make final concentration be 6-10g/L; adjusting the pH value to 5.8-6.0;
the callus induction culture medium is prepared by the following method:
adding 2,4-dichlorophenoxyacetic acid into MS culture medium to make final concentration 0.1-2.0mg/L, adding 6-benzylaminopurine to make final concentration 0.1-1.0mg/L, adding sucrose to make final concentration 20-30g/L, adding agar to make final concentration 6-10g/L; adjusting the pH value to 5.8-6.0;
the adventitious root induction culture medium is prepared by the following method:
adding indolebutyric acid to a final concentration of 0.5-2.0mg/L in an MS culture medium, adding sucrose to a final concentration of 20-30g/L, and adding agar to a final concentration of 6-10g/L; adjusting the pH value to 5.8-6.0;
the growth medium is prepared by the following method:
adding indolebutyric acid into MS culture medium to make final concentration be 2.0-6.0mg/L, adding kinetin to make final concentration be 0.2-0.6mg/L, adding sucrose to make final concentration be 20-30g/L; adjusting the pH value to 5.5-5.8.
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| CN111296288B (en) * | 2020-03-02 | 2023-03-24 | 内蒙古自治区生物技术研究院 | Culture method for inducing astragalus membranaceus callus and application of culture method |
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