CN111587785B - Culture method of hairy roots of astragalus membranaceus for promoting accumulation of flavonoid substances - Google Patents
Culture method of hairy roots of astragalus membranaceus for promoting accumulation of flavonoid substances Download PDFInfo
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Abstract
The invention provides a method for culturing hairy roots of astragalus membranaceus for promoting the accumulation of flavonoid substances, which comprises the steps of culturing aseptic seedlings of astragalus membranaceus, activating agrobacterium rhizogenes, co-culturing astragalus membranaceus explants and agrobacterium, and sterilizing, inducing and culturing and expanding the culturing. In order to promote the accumulation of flavonoid substances in hairy roots of astragalus, proper agrobacterium rhizogenes are selected to infect astragalus seedlings, different induction factors are applied at different stages of culture to cooperatively promote the accumulation of flavonoid substances, and the method specifically comprises the steps of applying CuCl2, agNO3, cdSe quantum dots and CdTe quantum dots at a co-culture stage, applying methyl jasmonate during the degerming culture process of hairy roots and applying blue light at an expansion culture stage.
Description
Technical Field
The invention relates to a method for culturing hairy roots of astragalus membranaceus for promoting the accumulation of flavonoid substances, and belongs to the technical field of astragalus membranaceus biotechnology culture.
Background
Astragalus mongholicus belongs to perennial herb plants of leguminosae, and is used as a root medicament, so far, more than 2000 years of history have been available. The astragalus has the main pharmacological effects of tonifying qi, strengthening exterior, promoting urination, expelling toxin, expelling pus, promoting granulation and the like, is mainly used for treating short breath, deficiency, palpitation, spontaneous perspiration, body deficiency edema, chronic nephritis, dehydration, chronic diarrhea, qi deficiency, qi and blood deficiency, sinking of middle-jiao, resisting bacteria, diminishing inflammation, reducing blood pressure, promoting urination, detoxifying, promoting bile flow and the like, and is a common Chinese herbal medicine.
Hairy root culture is a technology combining genetic engineering and cell engineering developed in the 80 th century, and is to integrate T-DNA contained in Ri plasmid of Agrobacterium rhizogenes into DNA of plant cells to induce the plant cells to generate hairy roots. Hairy root cultures have genetic and biochemical stability relative to cell cultures, and hairy roots can grow rapidly on hormone-free media and accumulate large amounts of secondary metabolites of higher economic value. Therefore, hairy roots are considered as excellent raw materials for obtaining secondary metabolites of plants. Hairy root culture has been considered in recent years as an important way to obtain useful secondary metabolites. It is of interest how to increase the accumulation of useful secondary metabolites in hairy roots during cultivation
Since the first successful transformation of higher plants with Agrobacterium rhizogenes in Ackermann 1997, more than 100 plant hairy root culture systems have been established to date, some of which are medicinal plants. For example, small-scale cultivation of hairy roots of Nicotiana tabacum, datura, belladonna, hyoscyamus, catharanthus roseus, lithospermum erythrorhizon, and Horseradish has been successful. The utilization of hairy root culture technology for large-scale production of effective secondary metabolites of medicinal plants has very attractive prospect, and has positive significance for protecting wild resources and ecological environment and realizing sustainable development.
Therefore, the invention hopes to further improve the induction culture method of the hairy roots of the astragalus so as to improve the induction efficiency of the hairy roots and promote the accumulation of flavonoid substances, and effectively solve the problems of lack of natural resources of the astragalus and shortage of market products.
Disclosure of Invention
The invention aims to overcome the problems of lack of natural resources of astragalus and shortage of related market products, and utilizes biotechnology to protect precious soil resources, and provides a method for culturing hairy roots of astragalus for promoting accumulation of flavonoid substances.
In order to achieve the above purpose, the invention provides a method for culturing hairy roots of astragalus membranaceus for promoting the accumulation of flavonoid substances, which comprises the following steps:
step 1, preparing the aseptic seedlings of astragalus
Soaking radix astragali seed in tap water for 20-24 hr, soaking in 70% ethanol for 10-15min, and washing with sterile water for 3-5 times; pouring 1% mercuric chloride solution into the container for soaking for 8-10min; flushing with sterile water for 3-5 times; inoculating to MS culture medium sterilized in advance, and culturing at 15-25deg.C with humidity of 50-60% to obtain radix astragali aseptic seedling;
step 2, activating and culturing strains
Adding Agrobacterium rhizogenes into liquid YEB culture medium, and culturing at 26-28deg.C and 220-250rpm until OD600 value is 0.5-0.6; then culturing the bacterial liquid in a 1/2MS liquid culture medium, and culturing at 150-180rpm for 20-30min at 26-28 ℃;
step 3, material preculture
Cutting cotyledon, stem, hypocotyl and petiole of aseptic seedling of radix astragali in step 1 into pieces of 0.4-0.5cm 2 Inoculating the small pieces serving as explants to MS culture medium added with 0.1-0.3mg/L plant hormone, and culturing for 24-48h in dark;
step 4. Co-culture of the explant and Agrobacterium rhizogenes
Fully contacting the explant in the step 3 with the bacterial liquid in the step 2 at 50-60rpm and 25-28 ℃ for 10-15 min; sucking the superfluous bacterial liquid on the surface by using sterile filter paper, and inoculating the superfluous bacterial liquid into an MS culture medium added with 0.2-0.4mg/L of induction factor for 2-4 days; the induction factor is CuCl 2 、AgNO 3 One or more than two of CdSe quantum dots and CdTe quantum dots;
step 5. Degerming induction culture of hairy roots
Washing the explant in the step 4 with sterile water, inoculating the explant to a 1/2MS solid culture medium containing 500mg/L cefotaxime sodium (cef), and culturing the explant in a dark place at 25 ℃; and changing a new culture medium at intervals of one week, and gradually reducing the concentration of cefotaxime sodium (cef);
separating the roots after the length reaches 3cm, placing the roots on a 1/2MS solid culture medium containing 50mg/L of cefotaxime sodium (cef) and 0.6-0.8mg/L of methyl jasmonate, carrying out successive transfer once a week, and culturing the roots on a 1/2MS solid culture medium without cefotaxime sodium (cef) after three successive transfers;
step 6, enlarged culture of hairy roots
Transferring the hairy roots in the step 5 into a 6,7-V liquid culture medium, performing expansion culture at 25-28 ℃ and 100-130rpm, applying blue light in the culture process, illuminating for 8-12h each day, and performing secondary culture every 18-20 days to obtain a large amount of growing hairy roots of astragalus.
Further, the astragalus seed is selected from one of astragalus mongholicus seed or astragalus membranaceus seed;
further, the composition per liter of the MS medium was as follows:
macroelements: KNO (KNO) 3 4-6mM，Ca(NO 3 ) 2 ·4H 2 O 4-6mM，MgSO 4 ·7H 2 O 1.5-2.5mM，KH 2 PO 4 0.8-1.2mM，
Trace elements: h 3 BO 3 44-48μM，MnCl 2 ·4H 2 O 8-10μM，ZnSO 4 ·7H 2 O 0.6-0.8μM，CuSO 4 ·5H 2 O 0.2-0.4μM，Na 2 MoO 4 ·2H 2 O 0.1-0.3μM，EDTA-Fe-Na 40-60μM；
Organic matter: 80-100 mu M inositol, 300-450 mu M sucrose and 300-500 mu M hydrolyzed casein.
Further, the composition of the 1/2MS culture medium is to halve the amount of macroelements in the MS culture medium; the 1/2MS solid culture medium contains 2-4% of agar.
Further, the agrobacterium strain is selected from one of ATCC15834, ATCC11325, ACCC10060, LBA9402, R1000, R1601, A4.
Further, the composition of the liquid YEB medium in step 2 is as follows: : taking 1g of yeast powder, 5g of beef extract, 5g of peptone, 5g of sucrose and MgSO 4 ·7H 2 Dissolving 0.5g of O in 1L of distilled water, adjusting the pH value to 7.4 by using sodium hydroxide, and sterilizing at 121 ℃ for 20min under high pressure;
further, the phytohormone in the step 3 is one of IBA and NAA.
Further, the composition and preparation method of the 6,7-V liquid medium in the step 6 are as follows:
A. preparation of component mother liquor
(1) Macroelement mother liquor: each liter of mother liquor contains 16g KNO 3 、2g(NH 4 ) 2 SO 4 、5g MgSO 4 ·7H 2 O、3g NaH 2 PO 4 、4g KCl、0.4g Na 2 HPO 4 ；
(2)CaCl 2 Mother liquor: each liter of mother liquor contains 4g CaCl 2 ·2H 2 O
(3) Trace element mother liquor: each liter of mother liquor contains 4g of MnSO 4 ·H 2 O、1.5g ZnSO 4 ·7H 2 O、5g H 3 BO 3 、0.05g KI、0.25g NaMoO 4 ·2H 2 O、0.25g CuSO 4 ·5H 2 O、0.25g CoCl 2 ·6H 2 O
(4) Ferric salt mother liquor: each liter of mother liquor contains 1.86g EDTA-Na 2 、1.39g FeSO 4 ·7H 2 O
(5) Vitamin mother liquor: each liter of mother liquor contains 0.125g of nicotinic acid, 0.05g of vitamin B1, 0.05g of vitamin B6 and 10g of inositol
B. The preparation method of the 6,7-V liquid culture medium comprises the following steps: collecting 50mL of macroelement mother solution and CaCl 2 50mL of mother solution, 1mL of microelement mother solution, 10mL of ferric salt mother solution, 10mL of vitamin mother solution and 30g of sucrose; the pH was adjusted to 5.8 by dissolving in 1L of distilled water.
Further, the step-down of the concentration of cefotaxime sodium (cef) in the step 4 is performed in a manner of 400mg/L, 300mg/L, 200mg/L, 100 mg/L.
The invention has the following beneficial effects:
in order to promote the accumulation of flavonoid substances in hairy roots of astragalus, proper agrobacterium rhizogenes is selected to infect astragalus seedlings, and different induction factors are applied at different stages of culture to synergistically promote the accumulation of flavonoid substances. Specifically, the induction factor CuCl is applied in the co-culture stage 2 、AgNO 3 The CdSe quantum dots and the CdTe quantum dots can promote the massive and rapid growth of hairy roots of astragalus, stimulate the secondary metabolic process of astragalus and improve the accumulation of flavonoid substances; the application of methyl jasmonate in the degerming culture process of hairy roots can also stimulate the secondary metabolic process of astragalus and improve the accumulation of flavonoid substances; and blue light is applied in the stage of expansion culture, so that the accumulation of flavonoid substances can be improved. In a word, the invention obtains the astragalus hairy roots with a large amount of flavonoid substances accumulated by adjusting the astragalus hairy root culture process, and effectively solves the problems of lack of natural resources of the astragalus and shortage of market products.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the following examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
1. The following raw materials are prepared
(1) Preparing an MS culture medium, wherein the composition of each liter of MS culture medium is as follows:
macroelements: KNO (KNO) 3 6mM，Ca(NO 3 ) 2 ·4H 2 O 6mM，MgSO 4 ·7H 2 O2.5mM，KH 2 PO 4 1.2mM，
Trace elements: h 3 BO 3 48μM，MnCl 2 ·4H 2 O 10μM，ZnSO 4 ·7H 2 O 0.6μM，CuSO 4 ·5H 2 O 0.2μM，Na 2 MoO 4 ·2H 2 O 0.3μM，EDTA-Fe-Na 60μM；
Organic matter: inositol 100. Mu.M, sucrose 450. Mu.M, hydrolyzed casein 500. Mu.M.
(2) Preparing a 1/2MS culture medium: reducing the dosage of macroelements in the MS culture medium by half to obtain a 1/2MS culture medium;
adding 3% of agar into the 1/2MS culture medium, and cooling to obtain a 1/2MS solid culture medium;
(3) The method for preparing the liquid YEB culture medium is as follows: taking 1g of yeast powder, 5g of beef extract, 5g of peptone, 5g of sucrose and MgSO 4 ·7H 2 Dissolving 0.5g of O in 1L of distilled water, adjusting the pH value to 7.4 by using sodium hydroxide, and sterilizing at 121 ℃ for 20min under high pressure;
(4) Preparing 4.6,7-V liquid culture medium:
A. preparation of component mother liquor
(1) Macroelement mother liquor: each liter of mother liquor contains 16g KNO 3 、2g(NH 4 ) 2 SO 4 、5g MgSO 4 ·7H 2 O、3g NaH 2 PO 4 、4g KCl、0.4g Na 2 HPO 4 ；
(2)CaCl 2 Mother liquor: each liter of mother liquor contains 4g CaCl 2 ·2H 2 O
(3) Trace element mother liquor: each liter of mother liquor contains 4g of MnSO 4 ·H 2 O、1.5g ZnSO 4 ·7H 2 O、5g H 3 BO 3 、0.05gKI、0.25g NaMoO 4 ·2H 2 O、0.25g CuSO 4 ·5H 2 O、0.25g CoCl 2 ·6H 2 O
(4) Ferric salt mother liquor: each liter of mother liquor contains 1.86g EDTA-Na 2 、1.39g FeSO 4 ·7H 2 O
(5) Vitamin mother liquor: each liter of mother liquor contains 0.125g of nicotinic acid, 0.05g of vitamin B1, 0.05g of vitamin B6 and 10g of inositol
The preparation method of the liquid culture medium B.6,7-V comprises the following steps: collecting 50mL of macroelement mother solution and CaCl 2 50mL of mother solution, 1mL of microelement mother solution, 10mL of ferric salt mother solution, 10mL of vitamin mother solution and 30g of sucrose; the pH was adjusted to 5.8 by dissolving in 1L of distilled water.
Example 1
A culture method for efficiently inducing hairy roots of astragalus comprises the following steps:
step 1, preparing the aseptic seedlings of astragalus
Soaking Mongolian astragalus seed in tap water for 24 hours, soaking in 70% ethanol for 15 minutes, and washing with sterile water for 5 times; pouring 1% mercuric chloride solution into the container for soaking for 10min; washing with sterile water for 5 times; inoculating to MS culture medium sterilized in advance, and culturing at 15deg.C with humidity of 60% to obtain radix astragali aseptic seedling;
step 2, activating and culturing strains
Agrobacterium rhizogenes LBA9402 was added to the liquid YEB medium and incubated at 28℃and 220rpm to an OD600 of 0.5; then, culturing the bacterial liquid in a 1/2MS liquid culture medium, and culturing for 20min at 180rpm at 28 ℃;
step 3, material preculture
Cutting cotyledon of aseptic seedling of radix astragali in step 1 into 0.4cm 2 Is inoculated as an explant to M supplemented with 0.3mg/L phytohormone IBACulturing in S culture medium in dark for 48 hr;
step 4. Co-culture of the explant and Agrobacterium rhizogenes
Fully contacting the explant in the step 3 with the bacterial liquid in the step 2 at 60rpm and 25 ℃ for 15 min; the surface redundant bacterial liquid is sucked by sterile filter paper and inoculated into an MS culture medium added with 0.4mg/L CdSe quantum dot for 4 days; step 5. Degerming induction culture of hairy roots
Washing the explant in the step 4 with sterile water, inoculating the explant to a 1/2MS solid culture medium containing 500mg/L cefotaxime sodium (cef), and culturing the explant in a dark place at 25 ℃; and changing a new culture medium at intervals of one week, and gradually reducing the concentration of cefotaxime sodium (cef);
separating the roots after the length reaches 3cm, placing the roots on a 1/2MS solid culture medium containing 50mg/L of cefotaxime sodium (cef) and 0.8mg/L of methyl jasmonate, carrying out successive substitution once a week, carrying out successive substitution for three times, and placing the roots on the 1/2MS solid culture medium without cefotaxime sodium (cef) for culture;
step 6, enlarged culture of hairy roots
Transferring the hairy roots in the step 5 into a 6,7-V liquid culture medium, performing expansion culture at 25 ℃ and 130rpm, applying blue light in the culture process, illuminating for 8 hours every day, and performing secondary transfer every 18 days to obtain a large amount of growing hairy roots of astragalus.
Example 2
A culture method for efficiently inducing hairy roots of astragalus comprises the following steps:
step 1, preparing the aseptic seedlings of astragalus
Soaking astragalus membranaceus seeds in tap water for 20 hours, soaking in 70% ethanol for 10 minutes, and washing with sterile water for 5 times; pouring 1% mercuric chloride solution into the container for soaking for 8min; washing with sterile water for 5 times; inoculating to MS culture medium sterilized in advance, and culturing at 25deg.C with humidity of 60% to obtain radix astragali aseptic seedling;
step 2, activating and culturing strains
Agrobacterium rhizogenes LBA9402 was added to the liquid YEB medium and incubated at 26℃and 220rpm to an OD600 of 0.6; then, culturing the bacterial liquid in a 1/2MS liquid culture medium, and culturing for 20min at 180rpm at 28 ℃;
step 3, material preculture
Cutting hypocotyl of aseptic seedling of radix astragali in step 1 into 0.4cm 2 Inoculating the small blocks serving as explants to MS culture medium added with 0.2mg/L phytohormone NAA, and culturing for 24 hours in dark place;
step 4. Co-culture of the explant and Agrobacterium rhizogenes
Fully contacting the explant in the step 3 with the bacterial liquid in the step 2 at 60rpm and 25 ℃ for 15 min; sucking the superfluous bacterial liquid on the surface by using sterile filter paper, and inoculating the superfluous bacterial liquid into an MS culture medium added with 0.3mg/L CdTe quantum dots for culturing for 4 days; step 5. Degerming induction culture of hairy roots
Washing the explant in the step 4 with sterile water, inoculating the explant to a 1/2MS solid culture medium containing 500mg/L cefotaxime sodium (cef), and culturing the explant in a dark place at 25 ℃; and changing a new culture medium at intervals of one week, and gradually reducing the concentration of cefotaxime sodium (cef);
separating the roots after the length reaches 3cm, placing the roots on a 1/2MS solid culture medium containing 50mg/L of cefotaxime sodium (cef) and 0.6/L of methyl jasmonate, carrying out successive substitution once a week for three times, and placing the roots on a 1/2MS solid culture medium without cefotaxime sodium (cef) for culture;
step 6, enlarged culture of hairy roots
Transferring the hairy roots in the step 5 into a 6,7-V liquid culture medium, performing expansion culture at 25 ℃ and 100rpm, applying blue light in the culture process, illuminating for 8 hours every day, and performing secondary transfer every 20 days to obtain a large amount of growing hairy roots of astragalus.
Example 3
A culture method for efficiently inducing hairy roots of astragalus comprises the following steps:
step 1, preparing the aseptic seedlings of astragalus
Soaking astragalus membranaceus seeds in tap water for 20 hours, soaking in 70% ethanol for 10 minutes, and washing with sterile water for 5 times; pouring 1% mercuric chloride solution into the container for soaking for 8min; washing with sterile water for 5 times; inoculating to MS culture medium sterilized in advance, and culturing at 25deg.C with humidity of 60% to obtain radix astragali aseptic seedling;
step 2, activating and culturing strains
Agrobacterium rhizogenes LBA9402 was added to the liquid YEB medium and incubated at 26℃and 220rpm to an OD600 of 0.6; then, culturing the bacterial liquid in a 1/2MS liquid culture medium, and culturing at 160rpm at 26 ℃ for 20min;
step 3, material preculture
Cutting cotyledon of aseptic seedling of radix astragali in step 1 into 0.4cm 2 Inoculating the small blocks serving as explants to MS culture medium added with 0.2mg/L phytohormone NAA, and culturing for 24 hours in dark place;
step 4. Co-culture of the explant and Agrobacterium rhizogenes
Fully contacting the explant in the step 3 with the bacterial liquid in the step 2 at 60rpm and 25 ℃ for 15 min; the surface excess bacterial liquid is sucked by sterile filter paper and inoculated to CuCl added with 0.3mg/L 2 Is cultured in MS culture medium for 3 days; step 5. Degerming induction culture of hairy roots
Washing the explant in the step 4 with sterile water, inoculating the explant to a 1/2MS solid culture medium containing 500mg/L cefotaxime sodium (cef), and culturing the explant in a dark place at 25 ℃; and changing a new culture medium at intervals of one week, and gradually reducing the concentration of cefotaxime sodium (cef);
separating the roots after the length reaches 3cm, placing the roots on a 1/2MS solid culture medium containing 50mg/L of cefotaxime sodium (cef) and 0.7mg/L of methyl jasmonate, carrying out successive substitution once a week, carrying out successive substitution for three times, and placing the roots on the 1/2MS solid culture medium without cefotaxime sodium (cef) for culture;
step 6, enlarged culture of hairy roots
Transferring the hairy roots in the step 5 into a 6,7-V liquid culture medium, performing expansion culture at 28 ℃ and 120rpm, applying blue light in the culture process, illuminating for 10 hours every day, and performing secondary transfer every 20 days to obtain a large amount of growing hairy roots of astragalus.
Example 4
A method for culturing hairy roots of radix astragali for promoting flavonoid accumulation comprises the following steps:
step 1, preparing the aseptic seedlings of astragalus
Soaking Mongolian astragalus seed in tap water for 22 hours, soaking in 70% ethanol for 14 minutes, and washing with sterile water for 4 times; pouring 1% mercuric chloride solution into the container for soaking for 9min; washing with sterile water for 4 times; inoculating to MS culture medium sterilized in advance, and culturing at 20deg.C with humidity of 55% to obtain radix astragali aseptic seedling;
step 2, activating and culturing strains
Agrobacterium rhizogenes was added to the liquid YEB medium and incubated at 27℃and 240rpm until an OD600 of 0.6 was reached; then, culturing the bacterial liquid in a 1/2MS liquid culture medium, and culturing for 20min at 180rpm at 27 ℃;
step 3, material preculture
Cutting the stem of aseptic seedling of radix astragali in step 1 into pieces with a size of 0.4cm 2 Inoculating the small blocks serving as explants to MS culture medium added with 0.1mg/L phytohormone IBA, and culturing for 36h in dark;
step 4. Co-culture of the explant and Agrobacterium rhizogenes
Fully contacting the explant in the step 3 with the bacterial liquid in the step 2 at 60rpm and 25 ℃ for 15 min; the surface excess bacterial liquid is sucked by sterile filter paper and inoculated to AgNO added with 0.4mg/L 3 Is cultured in MS culture medium for 3 days; step 5. Degerming induction culture of hairy roots
Washing the explant in the step 4 with sterile water, inoculating the explant to a 1/2MS solid culture medium containing 500mg/L cefotaxime sodium (cef), and culturing the explant in a dark place at 25 ℃; and changing a new culture medium at intervals of one week, and gradually reducing the concentration of cefotaxime sodium (cef);
separating the roots after the length reaches 3cm, placing the roots on a 1/2MS solid culture medium containing 50mg/L of cefotaxime sodium (cef) and 0.7mg/L of methyl jasmonate, carrying out successive substitution once a week, carrying out successive substitution for three times, and placing the roots on the 1/2MS solid culture medium without cefotaxime sodium (cef) for culture;
step 6, enlarged culture of hairy roots
Transferring the hairy roots in the step 5 into a 6,7-V liquid culture medium, performing expansion culture at 26 ℃ and 110rpm, applying blue light in the culture process, illuminating for 10 hours every day, and performing secondary transfer every 20 days to obtain a large amount of growing hairy roots of astragalus.
Comparative example 1
Step 4 was not applied with the inducer CdSe quantum dots, and the rest of the procedure was exactly the same as in example 1.
Comparative example 2
The 1/2MS medium described in step 5 was not supplemented with methyl jasmonate, and the rest of the procedure was identical to example 1.
Comparative example 3
In step 6, no blue light was applied, and the other steps were identical to those of example 1.
The invention adopts the following method to test the content of flavonoid substances in hairy roots of astragalus, and specifically comprises the following steps: drying and pulverizing radix astragali hairy roots in examples 1-4 and comparative examples 1-3, weighing 250mg of the sample, adding 25ml of 60% ethanol solution, reflux-extracting for 4h, cooling to room temperature, filtering, concentrating and drying the filtrate, dissolving with ethanol to 5ml, and passing through 0.22 μm membrane to be detected; the relevant experimental data are shown in table 1.
TABLE 1
As can be seen from the experimental data in Table 1, the present invention can enhance the accumulation of flavonoids in hairy roots by applying different induction factors in different culture stages of the hairy roots of Astragalus mongholicus, specifically, applying the induction factor CuCl in the co-culture stage 2 、AgNO 3 The CdSe quantum dots and the CdTe quantum dots can promote the massive and rapid growth of hairy roots of astragalus, stimulate the secondary metabolic process of astragalus and improve the accumulation of flavonoid substances; the application of methyl jasmonate in the degerming culture process of hairy roots can also stimulate the secondary metabolic process of astragalus and improve the accumulation of flavonoid substances; and blue light is applied in the stage of expansion culture, so that the accumulation of flavonoid substances can be improved.
It will be readily appreciated by those skilled in the art that the foregoing description is merely a preferred embodiment of the invention and is not intended to limit the invention, but any modifications, equivalents, improvements or alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (3)
1. A method for culturing hairy roots of astragalus membranaceus for promoting the accumulation of flavonoid substances is characterized by comprising the following steps of: the method comprises the following steps:
step 1, preparing the aseptic seedlings of astragalus
Soaking radix astragali seed in tap water for 20-24 hr, soaking in 70% ethanol for 10-15min, and washing with sterile water for 3-5 times; pouring 1% mercuric chloride solution into the container for soaking for 8-10min; flushing with sterile water for 3-5 times; inoculating to MS culture medium sterilized in advance, and culturing at 15-25deg.C with humidity of 50-60% to obtain radix astragali aseptic seedling; the astragalus seed is selected from one of astragalus mongholicus seeds or astragalus membranaceus seeds; the composition per liter of the MS medium was as follows:
macroelements: KNO (KNO) 3 4-6mM，Ca(NO 3 ) 2 ·4H 2 O 4-6mM，MgSO 4 ·7H 2 O 1.5-2.5mM，KH 2 PO 4 0.8-1.2mM，
Trace elements: h 3 BO 3 44-48μM，MnCl 2 ·4H 2 O 8-10μM，ZnSO 4 ·7H 2 O 0.6-0.8μM，CuSO 4 ·5H 2 O 0.2-0.4μM，Na 2 MoO 4 ·2H 2 O 0.1-0.3μM，EDTA-Fe-Na 40-60μM；
Organic matter: 80-100 mu M of inositol, 300-450 mu M of sucrose and 300-500 mu M of hydrolyzed casein;
step 2, activating and culturing strains
Adding Agrobacterium rhizogenes into liquid YEB culture medium, and culturing at 26-28deg.C and 220-250rpm until OD600 value is 0.5-0.6; then culturing the bacterial liquid in a 1/2MS liquid culture medium, and culturing at 150-180rpm for 20-30min at 26-28 ℃; the composition of the 1/2MS culture medium is to halve the dosage of macroelements in the MS culture medium; the 1/2MS solid culture medium contains 2-4% of agar;
step 3, material preculture
Cutting cotyledon, stem, hypocotyl and petiole of aseptic seedling of radix astragali in step 1 into pieces of 0.4-0.5cm 2 As explants of (C)Inoculating the body to MS culture medium added with 0.1-0.3mg/L plant hormone, and culturing in dark for 24-48 hr, wherein the plant hormone is one of IBA and NAA;
step 4. Co-culture of the explant and Agrobacterium rhizogenes
Fully contacting the explant in the step 3 with the bacterial liquid in the step 2 at 50-60rpm and 25-28 ℃ for 10-15 min; sucking the superfluous bacterial liquid on the surface by using sterile filter paper, and inoculating the superfluous bacterial liquid into an MS culture medium added with 0.2-0.4mg/L of induction factor for 2-4 days; the induction factor is CuCl 2 One or more than two of CdSe quantum dots and CdTe quantum dots; the agrobacterium strain is selected from one of ATCC15834, ATCC11325, ACCC10060, LBA9402, R1000, R1601, A4;
step 5. Degerming induction culture of hairy roots
Washing the explant in the step 4 with sterile water, inoculating the explant to a 1/2MS solid culture medium containing 500mg/L cefotaxime sodium (cef), and culturing the explant in a dark place at 25 ℃; and changing a new culture medium at intervals of one week, and gradually reducing the concentration of cefotaxime sodium (cef); gradually decreasing the concentration of cefotaxime sodium (cef) in a manner of 400mg/L, 300mg/L, 200mg/L, 100 mg/L;
separating the roots after the length reaches 3cm, placing the roots on a 1/2MS solid culture medium containing 50mg/L of cefotaxime sodium (cef) and 0.6-0.8mg/L of methyl jasmonate, carrying out successive transfer once a week, and culturing the roots on a 1/2MS solid culture medium without cefotaxime sodium (cef) after three successive transfers;
step 6, enlarged culture of hairy roots
Transferring the hairy roots in the step 5 into a 6,7-V liquid culture medium, performing expansion culture at 25-28 ℃ and 100-130rpm, applying blue light in the culture process, illuminating for 8-12h each day, and performing secondary culture every 18-20 days to obtain a large amount of growing hairy roots of astragalus.
2. The method for culturing hairy roots of astragalus membranaceus for promoting the accumulation of flavonoid substances according to claim 1, which is characterized in that: the composition of the liquid YEB medium in step 2 is as follows: taking 1g of yeast powder, 5g of beef extract, 5g of peptone, 5g of sucrose and MgSO 4 ·7H 2 O0.5 g was dissolved in 1L distilled water, adjusted to pH 7.4 with sodium hydroxide, and autoclaved for 20min at 121 ℃.
3. The method for culturing hairy roots of astragalus membranaceus for promoting the accumulation of flavonoid substances according to claim 1, which is characterized in that: the composition and preparation method of the 6,7-V liquid culture medium in the step 6 are as follows:
A. preparation of component mother liquor
(1) Macroelement mother liquor: each liter of mother liquor contains 16g KNO 3 、2g(NH 4 ) 2 SO 4 、5g MgSO 4 ·7H 2 O、3g NaH 2 PO 4 、4g KCl、0.4g Na 2 HPO 4 ；
(2) CaCl2 mother liquor: each liter of mother liquor contains 4g CaCl 2 ·2H 2 O
(3) Trace element mother liquor: each liter of mother liquor contains 4g of MnSO 4 ·H 2 O、1.5g ZnSO 4 ·7H 2 O、5g H 3 BO 3 、0.05g KI、0.25g NaMoO 4 ·2H 2 O、0.25g CuSO 4 ·5H 2 O、0.25g CoCl 2 ·6H 2 O
(4) Ferric salt mother liquor: each liter of mother liquor contains 1.86g EDTA-Na 2 、1.39g FeSO 4 ·7H 2 O
(5) Vitamin mother liquor: each liter of mother liquor contains 0.125g of nicotinic acid, 0.05g of vitamin B1, 0.05g of vitamin B6 and 10g of inositol
B. The preparation method of the 6,7-V liquid culture medium comprises the following steps: collecting 50mL of macroelement mother solution and CaCl 2 50mL of mother solution, 1mL of microelement mother solution, 10mL of ferric salt mother solution, 10mL of vitamin mother solution and 30g of sucrose; the pH was adjusted to 5.8 by dissolving in 1L of distilled water.
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