CN111587785A - Method for culturing astragalus membranaceus hairy roots for promoting accumulation of flavonoids - Google Patents
Method for culturing astragalus membranaceus hairy roots for promoting accumulation of flavonoids Download PDFInfo
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Abstract
The invention provides a method for culturing astragalus mongholicus hairy roots for promoting accumulation of flavonoids, which specifically comprises the steps of culturing astragalus mongholicus aseptic seedlings, activating agrobacterium rhizogenes, co-culturing astragalus mongholicus explants and agrobacterium rhizogenes, and performing sterilization induction culture and expansion culture. In order to promote accumulation of flavonoids in astragalus hairy roots, the method selects proper agrobacterium rhizogenes to infect astragalus seedlings, applies different induction factors to synergistically promote accumulation of flavonoids at different stages of culture, and specifically comprises the steps of applying CuCl2, AgNO3, CdSe quantum dots and CdTe quantum dots at a co-culture stage, applying methyl jasmonate in a degerming culture process of the hairy roots and applying blue light at an expansion culture stage.
Description
Technical Field
The invention relates to a method for culturing astragalus mongholicus hairy roots for promoting accumulation of flavonoids, and belongs to the technical field of astragalus mongholicus biotechnology cultivation.
Background
Astragalus root belongs to perennial herb of leguminosae, and has been used as medicine by root, so far, more than 2000 years of history. The astragalus root has the main pharmacological actions of tonifying qi, strengthening exterior, promoting urination, expelling toxin, discharging pus, promoting granulation and the like, is mainly used for treating short breath, collapse, palpitation, spontaneous perspiration, body weakness, edema, chronic nephritis, dehydration, chronic diarrhea, qi deficiency, deficiency of qi and blood, sinking of middle-jiao, has the effects of resisting bacteria, diminishing inflammation, reducing blood pressure, promoting urination, detoxifying and benefiting gallbladder 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 of the 20 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 produce hairy roots. The hairy root culture has genetic and biochemical stability compared with the cell culture, the hairy root can grow rapidly on a hormone-free culture medium and can accumulate a large amount of secondary metabolites with higher economic value. Thus, hairy roots are considered to be an excellent raw material for obtaining plant secondary metabolites. Hairy root culture has been considered in recent years as an important route to useful secondary metabolites. How to improve the accumulation capacity of useful secondary metabolites in hairy roots in the culture process becomes the focus of attention
Since the first successful transformation of higher plants with Agrobacterium rhizogenes in 1997 Ackermann, more than 100 plant hairy root culture systems have been established to date, many of which are medicinal plants. For example, small-scale culture of hairy roots of Nicotiana citrifolia, Datura stramonium, belladonna, hyoscyami, Vinca rosea, Lithospermum erythrorhizon, Horseradish has been successful. The method for producing the effective secondary metabolites of the medicinal plants in a large scale by utilizing the hairy root culture technology 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 astragalus mongholicus hairy roots so as to improve the induction efficiency of the hairy roots and promote the accumulation of flavonoids, and effectively solve the problems of the lack of natural resources of the astragalus mongholicus and the shortage of market products.
Disclosure of Invention
The invention aims to overcome the problems of the lack of natural resources of astragalus and the shortage of related market products, protect precious soil resources by utilizing biotechnology, and provide a culture method of astragalus hairy roots for promoting the accumulation of flavonoids.
In order to achieve the above object, the present invention provides a method for culturing astragalus membranaceus hairy roots for promoting accumulation of flavonoids, comprising the steps of:
step 1. preparation of Astragalus aseptic seedling
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; soaking in 1% mercuric chloride solution for 8-10 min; washing with sterile water for 3-5 times; inoculating to MS culture medium sterilized in advance, culturing at 15-25 deg.C and humidity of 50-60% to obtain sterile seedling of radix astragali;
step 2, strain activation and culture
Adding agrobacterium rhizogenes into a liquid YEB culture medium, culturing at 26-28 ℃ and 220-250rpm until the OD600 value is 0.5-0.6; then, culturing the bacterial liquid in 1/2MS liquid culture medium at the temperature of 26-28 ℃ and the rotation speed of 150-;
step 3 Material Pre-culture
Cutting cotyledon, stem, hypocotyl and petiole of the sterile radix astragali seedling in step 1 into pieces with size of 0.4-0.5cm2Inoculating the small blocks as explants to an MS culture medium added with 0.1-0.3mg/L phytohormone, and culturing for 24-48h in a dark place;
step 4. Co-culture of explant and Agrobacterium rhizogenes
Fully contacting the explant in the step 3 with the bacterial liquid in the step 2 for 10-15min at the speed of 50-60rpm and the temperature of 25-28 ℃; sucking the redundant bacteria liquid on the surface by using sterile filter paper, and inoculating the bacteria liquid into an MS culture medium added with 0.2-0.4mg/L of induction factor to culture for 2-4 days; the induction factor is one or more than two of CuCl2, AgNO3, CdSe quantum dots and CdTe quantum dots;
step 5, degerming induction culture of hairy roots
Washing the explant obtained in the step 4 with sterile water, and then inoculating the washed explant to 1/2MS solid culture medium containing 500mg/L cefotaxime sodium (cef) to be cultured in the dark at 25 ℃; replacing the culture medium with new one week at intervals, and gradually reducing the concentration of cefotaxime sodium (cef);
separating the roots after the roots reach 3cm, placing the roots on 1/2MS solid culture medium containing 50mg/L cefotaxime sodium (cef) and 0.6-0.8mg/L methyl jasmonate, carrying out subculture once every one week, and after three subcultures, placing the roots on 1/2MS solid culture medium without cefotaxime sodium (cef) for culture;
step 6, enlarged cultivation of hairy root
Transferring the hairy roots in the step 5 into a 6,7-V liquid culture medium, carrying out amplification culture at 25-28 ℃ and at 100-130rpm, applying blue light in the culture process, illuminating for 8-12h every day, and carrying out subculture once every 18-20 days to obtain a large amount of grown astragalus hairy roots.
Further, the astragalus seeds are selected from one of astragalus mongholicus seeds or astragalus membranaceus seeds;
further, the composition per liter of the MS medium is as follows:
macroelements: KNO34-6mM, Ca (NO3) 2.4H 2O 4-6mM, MgSO 4.7H 2O 1.5.5-2.5 mM, KH2PO40.8-1.2mM,
trace elements: h3BO344-48 μ M, MnCl 2.4H2O 8-10 μ M, ZnSO 4.7H2O 0.6-0.8 μ M, CuSO 4.5H2O 0.2-0.4 μ M, Na2MoO 4.2H2O 0.1-0.3 μ M, EDTA-Fe-Na 40-60 μ M;
organic matter: inositol 80-100. mu.M, sucrose 300-450. mu.M, and hydrolyzed casein 300-500. mu.M.
Further, the 1/2MS culture medium is composed by reducing the dosage of macroelements in the MS culture medium by half; the 1/2MS solid medium contains agar 2-4%.
Further, the agrobacterium strain is selected from one of ATCC15834, ATCC11325, ACCC10060, LBA9402, R1000, R1601, a 4.
Further, the composition of the liquid YEB medium in step 2 is as follows: : dissolving yeast powder 1g, beef extract 5g, peptone 5g, sucrose 5g, MgSO4 & 7H2O 0.5g in distilled water 1L, adjusting pH to 7.4 with sodium hydroxide, sterilizing at 121 deg.C under high pressure for 20 min;
further, the plant hormone in the step 3 is one of IBA and NAA.
Further, 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: the mother liquor contains 16g KNO3, 2g (NH4)2SO4, 5g MgSO4 & 7H2O, 3g NaH2PO4, 4g KCl and 0.4g Na2HPO4 per liter;
(2) CaCl2 mother liquor: the mother liquor contains 4g of CaCl2 & 2H2O per liter
(3) And (3) a microelement mother solution: the mother liquor contains 4g of MnSO4 & H2O, 1.5g of ZnSO4 & 7H2O, 5g H3BO3, 0.05g of KI, 0.25g of NaMoO4 & 2H2O, 0.25g of CuSO4 & 5H2O and 0.25g of CoCl2 & 6H2O per liter
(4) Mother liquor of iron salt: the mother liquor contains 1.86g of EDTA-Na2 and 1.39g of FeSO 4.7H 2O per liter
(5) Vitamin mother liquor: the mother liquor contains nicotinic acid 0.125g, vitamin B1 0.05g, vitamin B6 0.05g, and inositol 10g per liter
B. The preparation method of the 6,7-V liquid culture medium comprises the following steps: taking 50mL of macroelement mother liquor, 50mL of CaCl2 mother liquor, 1mL of microelement mother liquor, 10mL of ferric salt mother liquor, 10mL of vitamin mother liquor and 30g of cane sugar; dissolving in 1L distilled water to adjust pH to 5.8.
Further, the step-wise reduction of the concentration of cefotaxime sodium (cef) in the step 4 is performed according to the manners of 400mg/L, 300mg/L, 200mg/L and 100 mg/L.
The invention has the following beneficial effects:
in order to promote the accumulation of flavonoids in the hairy roots of astragalus, the invention selects proper agrobacterium rhizogenes to infect astragalus seedlings, and applies different induction factors to synergistically promote the accumulation of flavonoids at different stages of culture. Specifically, the induction factors CuCl2, AgNO3, CdSe quantum dots and CdTe quantum dots are applied in the co-culture stage, so that the massive and rapid growth of astragalus hairy roots can be promoted, the secondary metabolism process of astragalus is stimulated, and the accumulation of flavonoids is improved; the application of methyl jasmonate in the degerming culture process of the hairy roots can also stimulate the secondary metabolic process of the astragalus, so that the accumulation of flavonoids is improved; and blue light is applied in the stage of enlarged culture, so that the accumulation of flavonoids can be improved. In a word, the invention obtains the astragalus mongholicus hairy roots with a large amount of accumulated flavonoids by adjusting the astragalus mongholicus hairy root culture process, and effectively solves the problems of the lack of natural resources of astragalus mongholicus and the 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 is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
1. The following raw materials were prepared
(1) Preparing an MS culture medium, wherein the composition of each liter of MS culture medium is as follows:
macroelements: KNO 36 mM, Ca (NO3) 2.4H 2O 6mM, MgSO 4.7H 2O2.5mM, KH2PO41.2mM,
trace elements: h3BO 348 mu M, MnCl2 & 4H2O 10 mu M, ZnSO4 & 7H2O 0.6.6 mu M, CuSO4 & 5H2O0.2 mu M, Na2MoO4 & 2H2O 0.3 mu M, EDTA-Fe-Na 60 mu M;
organic matter: inositol 100. mu.M, sucrose 450. mu.M, and hydrolyzed casein 500. mu.M.
(2) Preparation of 1/2MS medium: reducing the dosage of macroelements in the MS culture medium by half to prepare 1/2MS culture medium;
adding 3% agar into 1/2MS culture medium, and cooling to obtain 1/2MS solid culture medium;
(3) the method of formulating liquid YEB medium is as follows: dissolving yeast powder 1g, beef extract 5g, peptone 5g, sucrose 5g, MgSO4 & 7H2O 0.5g in distilled water 1L, adjusting pH to 7.4 with sodium hydroxide, sterilizing at 121 deg.C under high pressure for 20 min;
(4) preparing a 4.6,7-V liquid culture medium:
A. preparation of component mother liquor
(1) Macroelement mother liquor: the mother liquor contains 16g KNO3, 2g (NH4)2SO4, 5g MgSO4 & 7H2O, 3g NaH2PO4, 4g KCl and 0.4g Na2HPO4 per liter;
(2) CaCl2 mother liquor: the mother liquor contains 4g of CaCl2 & 2H2O per liter
(3) And (3) a microelement mother solution: the mother liquor contains 4g of MnSO4 & H2O, 1.5g of ZnSO4 & 7H2O, 5g H3BO3, 0.05gKI, 0.25g of NaMoO4 & 2H2O, 0.25g of CuSO4 & 5H2O and 0.25g of CoCl2 & 6H2O per liter
(4) Mother liquor of iron salt: the mother liquor contains 1.86g of EDTA-Na2 and 1.39g of FeSO 4.7H 2O per liter
(5) Vitamin mother liquor: the mother liquor contains nicotinic acid 0.125g, vitamin B1 0.05g, vitamin B6 0.05g, and inositol 10g per liter
The preparation method of the B.6,7-V liquid culture medium comprises the following steps: taking 50mL of macroelement mother liquor, 50mL of CaCl2 mother liquor, 1mL of microelement mother liquor, 10mL of ferric salt mother liquor, 10mL of vitamin mother liquor and 30g of cane sugar; dissolving in 1L distilled water to adjust pH to 5.8.
Example 1
A culture method for efficiently inducing astragalus mongholicus hairy roots comprises the following steps:
step 1. preparation of Astragalus aseptic seedling
Soaking Mongolian radix astragali seed in tap water for 24 hr, soaking in 70% ethanol for 15min, and washing with sterile water for 5 times; soaking in 1% mercuric chloride solution for 10 min; washing with sterile water for 5 times; inoculating to MS culture medium sterilized in advance, culturing at 15 deg.C and humidity of 60% to obtain radix astragali aseptic seedling;
step 2, strain activation and culture
Adding agrobacterium rhizogenes LBA9402 into liquid YEB culture medium, culturing at 28 ℃ and 220rpm until OD600 value is 0.5; then, culturing the bacterial liquid in 1/2MS liquid culture medium, culturing for 20min at 28 ℃ and 180 rpm;
step 3 Material Pre-culture
Cutting cotyledon of the sterile seedling of radix astragali in step 1 into pieces with size of 0.4cm2Inoculating the small blocks as explants to an MS culture medium added with 0.3mg/L phytohormone IBA, and culturing for 48h in a dark place;
step 4. Co-culture of explant and Agrobacterium rhizogenes
Fully contacting the explant in the step 3 with the bacterial liquid in the step 2 for 15min at the conditions of 60rpm and 25 ℃; sucking the redundant bacteria liquid on the surface by using sterile filter paper, and inoculating the bacteria liquid into an MS culture medium added with 0.4mg/L CdSe quantum dots to culture for 4 days; step 5, degerming induction culture of hairy roots
Washing the explant obtained in the step 4 with sterile water, and then inoculating the washed explant to 1/2MS solid culture medium containing 500mg/L cefotaxime sodium (cef) to be cultured in the dark at 25 ℃; replacing the culture medium with new one week at intervals, and gradually reducing the concentration of cefotaxime sodium (cef);
separating the roots after the roots reach 3cm, placing the roots on 1/2MS solid culture medium containing 50mg/L cefotaxime sodium (cef) and 0.8mg/L methyl jasmonate, carrying out subculture once every other week and three times, and then placing the roots on 1/2MS solid culture medium without cefotaxime sodium (cef) for culture;
step 6, enlarged cultivation of hairy root
And (3) transferring the hairy roots in the step (5) into a 6,7-V liquid culture medium, carrying out amplification culture at 25 ℃ and 130rpm, applying blue light in the culture process, illuminating for 8h every day, and subculturing once every 18 days to obtain a large amount of growing astragalus hairy roots.
Example 2
A culture method for efficiently inducing astragalus mongholicus hairy roots comprises the following steps:
step 1. preparation of Astragalus aseptic seedling
Soaking Astragalus membranaceus seed in tap water for 20 hr, soaking in 70% ethanol for 10min, and washing with sterile water for 5 times; soaking in 1% mercuric chloride solution for 8 min; washing with sterile water for 5 times; inoculating to MS culture medium sterilized in advance, culturing at 25 deg.C and humidity of 60% to obtain radix astragali aseptic seedling;
step 2, strain activation and culture
Adding agrobacterium rhizogenes LBA9402 into liquid YEB culture medium, culturing at 26 ℃ and 220rpm until OD600 value is 0.6; then, culturing the bacterial liquid in 1/2MS liquid culture medium, culturing for 20min at 28 ℃ and 180 rpm;
step 3 Material Pre-culture
Cutting hypocotyl of the sterile seedling of radix astragali in step 1 into pieces with size of 0.4cm2Inoculating the small blocks as explants to an MS culture medium added with 0.2mg/L plant hormone NAA, and culturing for 24h in a dark place;
step 4. Co-culture of explant and Agrobacterium rhizogenes
Fully contacting the explant in the step 3 with the bacterial liquid in the step 2 for 15min at the conditions of 60rpm and 25 ℃; sucking the redundant bacteria liquid on the surface by using sterile filter paper, and inoculating the bacteria liquid into an MS culture medium added with 0.3mg/L CdTe quantum dots for culture for 4 days; step 5, degerming induction culture of hairy roots
Washing the explant obtained in the step 4 with sterile water, and then inoculating the washed explant to 1/2MS solid culture medium containing 500mg/L cefotaxime sodium (cef) to be cultured in the dark at 25 ℃; replacing the culture medium with new one week at intervals, and gradually reducing the concentration of cefotaxime sodium (cef);
separating the roots after the roots reach 3cm, placing the roots on 1/2MS solid culture medium containing 50mg/L cefotaxime sodium (cef) and 0.6/L methyl jasmonate, carrying out subculture once every other week and three times, and then placing the roots on 1/2MS solid culture medium without cefotaxime sodium (cef) for culture;
step 6, enlarged cultivation of hairy root
And (3) transferring the hairy roots in the step (5) into a 6,7-V liquid culture medium, carrying out amplification culture at 25 ℃ and 100rpm, applying blue light in the culture process, illuminating for 8h every day, and carrying out subculture once every 20 days to obtain a large amount of growing astragalus hairy roots.
Example 3
A culture method for efficiently inducing astragalus mongholicus hairy roots comprises the following steps:
step 1. preparation of Astragalus aseptic seedling
Soaking Astragalus membranaceus seed in tap water for 20 hr, soaking in 70% ethanol for 10min, and washing with sterile water for 5 times; soaking in 1% mercuric chloride solution for 8 min; washing with sterile water for 5 times; inoculating to MS culture medium sterilized in advance, culturing at 25 deg.C and humidity of 60% to obtain radix astragali aseptic seedling;
step 2, strain activation and culture
Adding agrobacterium rhizogenes LBA9402 into liquid YEB culture medium, culturing at 26 ℃ and 220rpm until OD600 value is 0.6; then, culturing the bacterial liquid in 1/2MS liquid culture medium, and culturing for 20min at 26 ℃ and 160 rpm;
step 3 Material Pre-culture
Cutting cotyledon of the sterile seedling of radix astragali in step 1 into pieces with size of 0.4cm2Inoculating the small blocks as explants to an MS culture medium added with 0.2mg/L plant hormone NAA, and culturing for 24h in a dark place;
step 4. Co-culture of explant and Agrobacterium rhizogenes
Fully contacting the explant in the step 3 with the bacterial liquid in the step 2 for 15min at the conditions of 60rpm and 25 ℃; sucking off the redundant bacteria liquid on the surface by using sterile filter paper, and inoculating the bacteria liquid into an MS culture medium added with 0.3mg/L CuCl2 to culture for 3 days; step 5, degerming induction culture of hairy roots
Washing the explant obtained in the step 4 with sterile water, and then inoculating the washed explant to 1/2MS solid culture medium containing 500mg/L cefotaxime sodium (cef) to be cultured in the dark at 25 ℃; replacing the culture medium with new one week at intervals, and gradually reducing the concentration of cefotaxime sodium (cef);
separating the roots after the roots reach 3cm, placing the roots on 1/2MS solid culture medium containing 50mg/L cefotaxime sodium (cef) and 0.7mg/L methyl jasmonate, carrying out subculture once every other week and three times, and then placing the roots on 1/2MS solid culture medium without cefotaxime sodium (cef) for culture;
step 6, enlarged cultivation of hairy root
And (3) transferring the hairy roots in the step (5) into a 6,7-V liquid culture medium, carrying out amplification culture at 28 ℃ and 120rpm, applying blue light in the culture process, illuminating for 10h every day, and carrying out subculture once every 20 days to obtain a large amount of growing astragalus hairy roots.
Example 4
A method for culturing astragalus membranaceus hairy roots for promoting accumulation of flavonoids comprises the following steps:
step 1. preparation of Astragalus aseptic seedling
Soaking Mongolian radix astragali seed in tap water for 22 hr, soaking in 70% ethanol for 14min, and washing with sterile water for 4 times; soaking in 1% mercuric chloride solution for 9 min; washing with sterile water for 4 times; inoculating to MS culture medium sterilized in advance, culturing at 20 deg.C and 55% humidity to obtain radix astragali aseptic seedling;
step 2, strain activation and culture
Adding agrobacterium rhizogenes into a liquid YEB culture medium, and culturing at 27 ℃ and 240rpm until the OD600 value is 0.6; then, culturing the bacterial liquid in 1/2MS liquid culture medium, culturing for 20min at 27 ℃ and 180 rpm;
step 3 Material Pre-culture
Cutting the petioles of the astragalus membranaceus aseptic seedlings in the step 1 into 0.4cm in size2Inoculating the small blocks as explants to an MS culture medium added with 0.1mg/L phytohormone IBA, and culturing for 36h in a dark place;
step 4. Co-culture of explant and Agrobacterium rhizogenes
Fully contacting the explant in the step 3 with the bacterial liquid in the step 2 for 15min at the conditions of 60rpm and 25 ℃; sucking off the redundant bacteria liquid on the surface by using sterile filter paper, and inoculating the bacteria liquid into an MS culture medium added with 0.4mg/L AgNO3 to culture for 3 days; step 5. degerming induction culture of hairy root
Washing the explant obtained in the step 4 with sterile water, and then inoculating the washed explant to 1/2MS solid culture medium containing 500mg/L cefotaxime sodium (cef) to be cultured in the dark at 25 ℃; replacing the culture medium with new one week at intervals, and gradually reducing the concentration of cefotaxime sodium (cef);
separating the roots after the roots reach 3cm, placing the roots on 1/2MS solid culture medium containing 50mg/L cefotaxime sodium (cef) and 0.7mg/L methyl jasmonate, carrying out subculture once every other week and three times, and then placing the roots on 1/2MS solid culture medium without cefotaxime sodium (cef) for culture;
step 6, enlarged cultivation of hairy root
And (3) transferring the hairy roots in the step (5) into a 6,7-V liquid culture medium, carrying out amplification culture at 26 ℃ and 110rpm, applying blue light in the culture process, illuminating for 10h every day, and carrying out subculture once every 20 days to obtain a large amount of growing astragalus hairy roots.
Comparative example 1
Step 4 no induction factor CdSe quantum dots were applied, and the rest of the procedure was exactly the same as in example 1.
Comparative example 2
The 1/2MS medium in step 5 was supplemented with no methyl jasmonate, and the rest of the procedure was exactly the same as in example 1.
Comparative example 3
No blue light was applied in step 6 and the rest of the procedure was exactly the same as in example 1.
The invention adopts the following method to test the content of the flavonoid substances in the astragalus hairy roots, and the method specifically comprises the following steps: drying and crushing the hairy roots of the astragalus mongholicus in the examples 1-4 and the comparative examples 1-3, weighing 250mg of a sample, adding 25ml of a 60% ethanol solution, refluxing and extracting for 4 hours, cooling to room temperature, filtering, concentrating and drying filtrate, dissolving with ethanol to a constant volume of 5ml, and passing through a 0.22 mu m membrane to be detected; the relevant experimental data are shown in table 1.
TABLE 1
According to the experimental data in table 1, the invention can find that the accumulation of flavonoids in the hairy roots can be improved by applying different induction factors at different culture stages of the astragalus hairy roots, and specifically, the application of the induction factors CuCl2, AgNO3, CdSe quantum dots and CdTe quantum dots at the co-culture stage can promote the large and rapid growth of the astragalus hairy roots, stimulate the secondary metabolism process of the astragalus and improve the accumulation of flavonoids; the application of methyl jasmonate in the degerming culture process of the hairy roots can also stimulate the secondary metabolic process of the astragalus, so that the accumulation of flavonoids is improved; and blue light is applied in the stage of enlarged culture, so that the accumulation of flavonoids can be improved.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (9)
1. A method for culturing astragalus membranaceus hairy roots for promoting accumulation of flavonoids is characterized by comprising the following steps: the method comprises the following steps:
step 1. preparation of Astragalus aseptic seedling
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; soaking in 1% mercuric chloride solution for 8-10 min; washing with sterile water for 3-5 times; inoculating to MS culture medium sterilized in advance, culturing at 15-25 deg.C and humidity of 50-60% to obtain sterile seedling of radix astragali;
step 2, strain activation and culture
Adding agrobacterium rhizogenes into a liquid YEB culture medium, culturing at 26-28 ℃ and 220-250rpm until the OD600 value is 0.5-0.6; then, culturing the bacterial liquid in 1/2MS liquid culture medium at the temperature of 26-28 ℃ and the rotation speed of 150-;
step 3 Material Pre-culture
Cutting cotyledon, stem, hypocotyl and petiole of the sterile radix astragali seedling in step 1 into pieces with size of 0.4-0.5cm2Inoculating the small blocks as explants to an MS culture medium added with 0.1-0.3mg/L phytohormone, and culturing for 24-48h in a dark place;
step 4. Co-culture of explant and Agrobacterium rhizogenes
Fully contacting the explant in the step 3 with the bacterial liquid in the step 2 for 10-15min at the speed of 50-60rpm and the temperature of 25-28 ℃; sucking the redundant bacteria liquid on the surface by using sterile filter paper, and inoculating the bacteria liquid into an MS culture medium added with 0.2-0.4mg/L of induction factor to culture for 2-4 days; the induction factor is one or more than two of CuCl2, AgNO3, CdSe quantum dots and CdTe quantum dots;
step 5, degerming induction culture of hairy roots
Washing the explant obtained in the step 4 with sterile water, and then inoculating the washed explant to 1/2MS solid culture medium containing 500mg/L cefotaxime sodium (cef) to be cultured in the dark at 25 ℃; replacing the culture medium with new one week at intervals, and gradually reducing the concentration of cefotaxime sodium (cef);
separating the roots after the roots reach 3cm, placing the roots on 1/2MS solid culture medium containing 50mg/L cefotaxime sodium (cef) and 0.6-0.8mg/L methyl jasmonate, carrying out subculture once every one week, and after three subcultures, placing the roots on 1/2MS solid culture medium without cefotaxime sodium (cef) for culture;
step 6, enlarged cultivation of hairy root
Transferring the hairy roots in the step 5 into a 6,7-V liquid culture medium, carrying out amplification culture at 25-28 ℃ and at 100-130rpm, applying blue light in the culture process, illuminating for 8-12h every day, and carrying out subculture once every 18-20 days to obtain a large amount of grown astragalus hairy roots.
2. The method for culturing astragalus membranaceus hairy roots capable of promoting accumulation of flavonoids as claimed in claim 1, wherein the culture method comprises the following steps: the radix astragali seed is selected from one of Mongolian radix astragali seed or membrane pod radix astragali seed.
3. The method for culturing the hairy roots of astragalus membranaceus for promoting accumulation of flavonoids according to any one of claims 1 to 2, wherein the method comprises the following steps: the composition per liter of the MS medium was as follows:
macroelements: KNO34-6mM, Ca (NO3) 2.4H 2O 4-6mM, MgSO 4.7H 2O 1.5.5-2.5 mM, KH2PO40.8-1.2mM,
trace elements: h3BO344-48 μ M, MnCl 2.4H2O 8-10 μ M, ZnSO 4.7H2O 0.6-0.8 μ M, CuSO 4.5H2O 0.2-0.4 μ M, Na2MoO 4.2H2O 0.1-0.3 μ M, EDTA-Fe-Na 40-60 μ M;
organic matter: inositol 80-100. mu.M, sucrose 300-450. mu.M, and hydrolyzed casein 300-500. mu.M.
4. The method for culturing the hairy roots of astragalus membranaceus for promoting accumulation of flavonoids according to any one of claims 1 to 3, wherein the method comprises the following steps: the 1/2MS culture medium is composed by halving the dosage of macroelements in the MS culture medium; the 1/2MS solid medium contains agar 2-4%.
5. The method for culturing the hairy roots of astragalus membranaceus for promoting accumulation of flavonoids according to any one of claims 1 to 4, wherein the method comprises the following steps: the agrobacterium strain is selected from one of ATCC15834, ATCC11325, ACCC10060, LBA9402, R1000, R1601 and A4.
6. The method for culturing the hairy roots of astragalus membranaceus for promoting accumulation of flavonoids according to any one of claims 1 to 5, wherein the method comprises the following steps: the composition of the liquid YEB medium in step 2 is as follows: dissolving yeast powder 1g, beef extract 5g, peptone 5g, sucrose 5g, MgSO4 & 7H2O 0.5g in 1L distilled water, adjusting pH to 7.4 with sodium hydroxide, sterilizing at 121 deg.C under high pressure for 20 min.
7. The method for culturing the hairy roots of astragalus membranaceus for promoting accumulation of flavonoids according to any one of claims 1 to 5, wherein the method comprises the following steps: the plant hormone in the step 3 is one of IBA and NAA.
8. The method for culturing the hairy roots of astragalus membranaceus for promoting accumulation of flavonoids according to any one of claims 1 to 5, wherein the method comprises the following steps: 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: the mother liquor contains 16g KNO3, 2g (NH4)2SO4, 5g MgSO4 & 7H2O, 3g NaH2PO4, 4g KCl and 0.4g Na2HPO4 per liter;
(2) CaCl2 mother liquor: the mother liquor contains 4g of CaCl2 & 2H2O per liter
(3) And (3) a microelement mother solution: the mother liquor contains 4g of MnSO4 & H2O, 1.5g of ZnSO4 & 7H2O, 5g H3BO3, 0.05gKI, 0.25g of NaMoO4 & 2H2O, 0.25g of CuSO4 & 5H2O and 0.25g of CoCl2 & 6H2O per liter
(4) Mother liquor of iron salt: the mother liquor contains 1.86g of EDTA-Na2 and 1.39g of FeSO 4.7H 2O per liter
(5) Vitamin mother liquor: the mother liquor contains nicotinic acid 0.125g, vitamin B1 0.05g, vitamin B6 0.05g, and inositol 10g per liter
B. The preparation method of the 6,7-V liquid culture medium comprises the following steps: taking 50mL of macroelement mother liquor, 50mL of CaCl2 mother liquor, 1mL of microelement mother liquor, 10mL of ferric salt mother liquor, 10mL of vitamin mother liquor and 30g of cane sugar; dissolving in 1L distilled water to adjust pH to 5.8.
9. The method for culturing the hairy roots of astragalus membranaceus for promoting accumulation of flavonoids according to any one of claims 1 to 5, wherein the method comprises the following steps: the step-wise reduction of the concentration of cefotaxime sodium (cef) in the step 4 is performed according to the modes of 400mg/L, 300mg/L, 200mg/L and 100 mg/L.
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