CN113897383B - Induction and culture method for hairy root system of ammopiptanthus mongolicus for improving formononetin yield - Google Patents
Induction and culture method for hairy root system of ammopiptanthus mongolicus for improving formononetin yield Download PDFInfo
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Abstract
The invention relates to the technical field of genetic engineering, in particular to an induction and culture method of hairy root system of ammopiptanthus mongolicus, which can improve the yield of formononetin. The method comprises the steps of (1) obtaining an ammopiptanthus mongolicus explant; (2) selecting and activating agrobacterium; (3) secondary activation of agrobacterium; (4) infecting the explant with agrobacterium; (5) induction and propagation of hairy roots; (6) carrying out amplification culture; by the method, hairy roots capable of producing formononetin can be obtained; the content of formononetin produced by the hairy roots is obviously improved and is improved by 14 times compared with that of wild plants; the hairy roots of the Mongolian ammopiptanthus mongolicus have the advantages of quick growth, short period, no limitation of seasons, high relative yield of secondary metabolites and the like, and can be used for industrially producing the formononetin of the Mongolian ammopiptanthus mongolicus.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to an induction and culture method of hairy root system of ammopiptanthus mongolicus, which can improve the yield of formononetin.
Background
Ammopiptanthus mongolicus (Maxim. ex kon) Chengf.) also called Mongolia ammopiptanthus mongolicus and ammopiptanthus latifolia, belongs to the genus Ammopiptanthus in the subfamily Papilionaceae of Leguminosae, is the only super-arid evergreen broadleaf shrub species in the desert area of China, and is listed in the 1 st collection of rare or endangered protective plants by the nation. According to records in traditional Chinese medicine dictionary, national Chinese herbal medicine compilation and inner Mongolia plant medicine record, the ammopiptanthus mongolicus is a traditional national medicinal material (Mongolian medicine) named Meng and Harragaga, branches and leaves of the ammopium mongolicum regel can be used as medicines, and the ammopium mongolicum regel has the effects of dispelling wind-damp, promoting blood circulation, removing blood stasis and relieving pain, can be used for treating chilblain by external application, and particularly has a remarkable effect of treating rheumatic arthritis. Research shows that the ethanol extract of the ammopiptanthus mongolicus has good in-vivo blood sugar reducing activity, the anticancer substance JA1 extracted from ammopiptanthus mongolicus seeds has obvious inhibition effect on mouse tumor and human hepatoma cells, the ammopiptanthus mongolicus stem extract can inhibit the growth of diamondback moth larvae, and 1g/L and above of the water extract of the overground part of the ammopiptanthus mongolicus has good insecticidal effect on pine wood nematodes. The chemical components of the ammopiptanthus mongolicus are relatively complex, and the compounds separated and extracted from the ammopiptanthus mongolicus mainly comprise alkaloids, flavone, isoflavone, organic acid, stilbenes, sterols, monoterpenes and the like, and have the effects of resisting tumors, viruses, bacteria, oxidation, pests and the like. However, the ammopiptanthus mongolicus grows slowly under natural conditions, the survival rate of seedlings is very low, the natural updating capability is lacked, the regeneration force of transplanted seedling roots is weak, and the precious desert tree species are rare day by day and difficult to exert the advantages of the ammopiptanthus mongolicus due to artificial grazing and firewood chopping damage. Therefore, from the viewpoint of protecting endangered species and further exploring the precious resources of the species, researches on germplasm resource conservation, propagation expansion and innovation, development and utilization of secondary metabolites, functional gene mining, proteome, metabolome and the like of the ammopiptanthus mongolicus systematically are imperative.
Hairy roots (Hairy roots) are phenotypes in which a plant or an organ or tissue is infected with Agrobacterium rhizogenes to produce a large number of Hairy roots. The hairy root system has the characteristics of hormone autotrophy, strong genetic stability, high growth speed, strong secondary metabolism synthesis capability and the like, is an effective way for producing the secondary metabolites of medicinal plants, and provides possibility for realizing the industrial production of the secondary metabolites; in addition, the hairy roots have the characteristic of autonomous growth, can be induced into regenerated plants, can keep genetic stability in progeny regeneration, and provide support for molecular improvement breeding. Usually, the secondary metabolites synthesized by the parent plant can be produced by using hairy roots, and the yield of the secondary metabolites is often higher than the amount synthesized by the plant itself. The method for producing the active substance by utilizing the hairy root bioreactor can not only occupy the land but also protect the wild resources, and is an effective way for the sustainable development of endangered resources.
Agrobacterium rhizogenes is a soil bacterium with a very broad host range and is capable of infecting almost all dicotyledonous plants and a few monocotyledonous plants. Ri plasmid of agrobacterium rhizogenes is widely applied to plant genetic engineering, production of plant secondary metabolites, plant variety improvement and plant cultivation. The hairy roots generated by the transformed plants have the following advantages: 1) the hairy roots have the advantages of rapid growth and hormone autonomy, 2) the hairy roots can be conveniently transferred into foreign genes, 3) the frequency of the hairy roots of Ri plasmid transformed plants for differentiating normal plants is high, the ploidy is stable, a series is easily established, 4) the clone has a parent characteristic secondary metabolic pathway, the production capacity of secondary metabolites is high, the content is stable, and 5) the hairy roots are from a single cell, thereby providing great convenience for screening excellent lines.
At present, the induction and culture method of the hairy root is researched in many plants, for example, the invention patent (CN201910261837.4) discloses an induction method of the warm yam transgenic hairy root, and the method adopts a conventional agrobacterium activation method, establishes a warm yam transgenic hairy root induction system and realizes the rapid and effective propagation of the warm yam. Especially in medicinal plants, the induction and culture methods of the hairy root are more widely researched, for example, a high-efficiency induction method (CN109511553A) of the golden thread hairy root is disclosed, a high-efficiency induction system of the golden thread hairy root is firstly established, the induction rate of the hairy root is as high as 88.7 percent, and the method provides convenience for medicinal secondary metabolites. However, studies of ammopiptanthus mongolicus by scholars at home and abroad mainly focus on chemical components and pharmacological activity, for example, the invention patent (CN201711297833.9) provides a propagation method of ammopiptanthus mongolicus tissue culture seedlings, the method collects explants from aseptic seedlings to propagate the tissue culture seedlings, and the method has the advantages of simple operation, short time, zero pollution rate, high emergence rate and the like. There is no research on the induction and culture method of ammopiptanthus mongolicus hairy roots, and no report on the strain activation method in the hairy root culture process.
The inventor surprisingly discovers that in the process of inducing and culturing hairy roots of mongolian ammopiptanthus mongolicus, the inducer disclosed by the invention is used for carrying out secondary activation on agrobacterium rhizogenes, the activated agrobacterium rhizogenes is used for inducing the hairy roots, the yield of the generated Mongolian ammopiptanthus mongolicus for producing the formononetin is remarkably improved, the method is a very effective biological production method for obtaining the Mongolian ammopiptanthus mongolicus, and the endangered resource of the Mongolian ammopiptanthus mongolicus can be protected.
Disclosure of Invention
Aiming at the technical problem, the invention provides a method for inducing and culturing hairy root systems of ammopiptanthus mongolicus, which is used for improving the yield of formononetin, and the culture method comprises the following steps:
(1) obtaining an ammopiptanthus mongolicus explant: selecting mongolian ammopiptanthus mongolicus seeds, carrying out surface disinfection treatment, placing the disinfected seeds in a germination culture medium to obtain aseptic seedlings, and selecting different explant parts for a subsequent hairy root induction experiment according to the experiment;
(2) agrobacterium selection and activation: selecting an agrobacterium strain, picking a monoclonal from a culture plate, placing the monoclonal in 5mL of LB liquid medium containing corresponding antibiotics, and carrying out shaking culture at 28 ℃ for 20h at 200 r/min; taking 5mL of the above bacterial solution, inoculating into 50mL LB liquid medium for amplification culture, performing shaking culture at 28 deg.C and 240r/min to make OD of bacterial solution600About 1.5;
(3) and (3) secondary activation of agrobacterium: step (2)) Centrifuging the obtained Agrobacterium liquid at 5000 × g for 10min, collecting thallus, resuspending and diluting thallus with inducer to obtain infected bacterial liquid OD600Activating for 10-40min with inducer at appropriate infection concentration of 0.4-1.0 to obtain infection solution;
(4) infecting the explant with agrobacterium: treating an ammopiptanthus mongolicus explant, and infecting the treated ammopiptanthus mongolicus explant by using the infection liquid obtained in the step (3);
(5) hairy root induction and propagation: placing the infected explants in an accelerant for co-culture for 3-5 d; the growth medium containing antibiotics can be placed in 12-18 days, and hairy roots can be generated;
(6) and (3) amplification culture: cutting off from the explant when the hairy root of ammopiptanthus mongolicus grows to 1-3cm, putting the cut hairy root into a solid 1/2MS culture medium for in vitro rejuvenation and growth, and putting the hairy root into a liquid B5 culture medium for in vitro amplification culture when the hairy root grows to 4-5 cm;
the inducer in the step (3) is a liquid 1/2MS culture medium containing 100-500 mu mol/L acetosyringone, and the mass fraction of sucrose in the liquid 1/2MS culture medium is 5%; the time for infecting the explants in the step (4) is 10-30 min; the accelerant in the step (5) is a solid 1/2MS culture medium containing 100 mu mol/L-500 mu mol/L acetosyringone, and the mass fraction of sucrose in the solid 1/2MS culture medium is 5%.
Preferably, the inducer in the step (3) is a liquid 1/2MS culture medium containing 300 mu mol/L acetosyringone, and the mass fraction of sucrose in the liquid 1/2MS culture medium is 5%.
Preferably, the promoter in the step (5) is solid 1/2MS culture medium containing 100 μmol/L acetosyringone, and the mass fraction of sucrose in the solid 1/2MS culture medium is 5%.
Preferably, the infected explants are put into an accelerant to be co-cultured for 4d in the step (5); the growth medium 12-14d containing antibiotics can have hairy root;
preferably, the ammopiptanthus mongolicus explant in the step (1) comprises an epicotyl, a hypocotyl, cotyledons, true leaves, stems and roots.
Preferably, the ammopiptanthus mongolicus explant in the step (1) is a cotyledon.
Preferably, the time for infecting the explant in step (4) is 15 min.
Preferably, the method for treating ammopiptanthus mongolicus explants in step (4) comprises the following steps: pricking holes on the surface of the explant, scribing and cutting.
Preferably, the hole pricking on the surface of the explant comprises a hole which pricks through the explant and a hole which does not prick through the explant.
Preferably, the hole pricking on the surface of the explant is a hole which does not prick through the explant.
Preferably, the agrobacterium in step (2) is any one of agrobacterium a4, agrobacterium C58C1, agrobacterium ATCC15834, agrobacterium K599, agrobacterium LBA9402 and agrobacterium R1601.
Preferably, the agrobacterium in step (2) is agrobacterium a 4.
Preferably, 10, the method for inducing and culturing hairy root system of ammopiptanthus mongolicus for improving formononetin yield is applied to preparation of the ammopiptanthus mongolicus.
The invention has the beneficial effects that: the invention provides a novel inducer, wherein the inducer is a liquid 1/2MS culture medium containing 100 mu mol/L-500 mu mol/L acetosyringone, and the mass fraction of sucrose in the liquid 1/2MS culture medium is 5 percent and is used for secondary activation of agrobacterium; compared with the traditional hairy root induction and culture method, the method adds an inducer secondary activation strain; the invention provides an inducing and culturing method of hairy root system of mongolian ammopiptanthus mongolicus for improving the yield of formononetin, and hairy roots of the formononetin with high yield can be obtained by the method; fourthly, infecting mongolian ammopiptanthus mongolicus by using agrobacterium rhizogenes to induce hairy root tissue to grow, establishing an in-vitro hairy root culture system, and promoting the content of formononetin of the mongolian ammopiptanthus mongolicus to be remarkably improved by utilizing the vigorous growth power of the hairy root tissue and the efficient biosynthesis capacity of the hairy root tissue on secondary metabolites and to be improved by 14 times compared with wild plants; the hairy roots of the mongolian ammopiptanthus mongolicus have the advantages of quick growth, short period, no limitation of seasons, high relative yield of secondary metabolites and the like, and can be used for industrially producing the formononetin of the mongolian ammopiptanthus mongolicus. Provides an available way for the industrial production of the secondary metabolite of the mongolian ammopiptanthus mongolicus by utilizing hairy roots in the future, and is beneficial to the protection and sustainable utilization of the endangered resource plants on the other hand.
Drawings
FIG. 1 sterile seedlings of Mongolian ammopiptanthus mongolicus
FIG. 2 Induction and growth of Ammopiptanthus mongolicus explants
b: mongolian sand Chinese ilex leaf implant
c: induction of hairy root of ammopiptanthus mongolicus
d: hairy root elongation growth of Mongolian ammopiptanthus mongolicus
FIG. 3 the growing process of hairy roots of Mongolian ammopiptanthus mongolicus
e: in-vitro rejuvenation culture of hairy roots of Mongolian holly
f: in vitro proliferation and growth of hairy root of Mongolian holly
g: hairy root tissue of Mongolian ammopiptanthus mongolicus growing for 60d
Detailed Description
The technical solution of the present invention is described in detail below with reference to specific examples, but it should be noted that the scope of the present invention is not limited to the following examples. In the following examples of the present invention, the culture media used include 1/2MS medium and B5 medium, which are both conventionally used by those skilled in the art, and were obtained from reagent companies by the inventors in the present experiment.
In the following examples of the present invention, the sources of the strains were:
agrobacterium a 4: purchased from China agricultural microorganism strain preservation management center and having the self-numbering ACCC10060
Agrobacterium ATCC 15834: purchased from American model culture Collection Bank
Agrobacterium C58C 1: laboratory preservation of Chuguo professor of Chuguo of Zhejiang university of traditional Chinese medicine
Agrobacterium K599: this laboratory preserves
Agrobacterium LBA 9402: this laboratory preserves
Agrobacterium R1601: this laboratory preserves
In the following examples of the invention, the sources of reagents used are disclosed below:
acetosyringone: purchased from Sigma Co
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the subject matter herein.
Formononetin is a flavonoid compound and exists in various plants such as astragalus, suberect spatholobus stem and the like. Formononetin has anticancer effect, and can be used for preventing and treating breast cancer, prostate and colon cancer. Experts think that formononetin can reduce indexes such as matrix metalloproteinase, endostatin, basic fibroblast growth factor and the like in pleural fluid and serum of elderly patients with advanced lung cancer and the level of tumor markers, and has obvious clinical effect. Meanwhile, the medicine can improve the life quality of patients and has important significance for clinically treating the advanced lung cancer of the elderly.
The invention relates to a method for pricking holes on the surface of an external implant, which comprises the following steps: piercing fine holes in the surface of the implant using a sharp member such as a needle, knife, etc.;
the term "hole through the explant" as used herein means that the sharp member extends into the explant and penetrates the explant.
The term "hole without piercing the explant" as used herein means that the sharp member extends into the interior of the explant but does not penetrate the explant.
The scribing in the invention refers to scribing on the surface of the explant.
The term "cutting" as used herein refers to the cutting of explants into small pieces.
The browning is a phenomenon that in the process of inducing primary differentiation or redifferentiation of an explant in plant tissue culture, self tissues release brown substances from the surface to a culture medium so that the culture medium gradually turns brown, and the explant is further browned and dies.
Example one method for inducing and culturing hairy root line of ammopiptanthus mongolicus to increase formononetin yield
1. Experimental methods
(1) Obtaining an ammopiptanthus mongolicus explant: and (4) selecting full and consistent mongolian ammopiptanthus mongolicus seeds, and carrying out surface disinfection treatment. Placing the sterilized seeds in a germination culture medium to obtain sterile seedlings, and selecting six different explant parts of epicotyl, hypocotyl, cotyledon, true leaf, stem and root for a subsequent hairy root induction experiment according to the experiment as shown in figure 1;
(2) agrobacterium selection and activation: selecting an agrobacterium strain, picking a monoclonal from a culture plate, placing the monoclonal in 5mL of LB liquid medium containing corresponding antibiotics, and carrying out shaking culture at 28 ℃ for 20h at 200 r/min; taking 5mL of the above bacterial solution, inoculating into 50mL LB liquid medium for amplification culture, performing shaking culture at 28 deg.C and 240r/min to make OD of bacterial solution600About 1.5;
the agrobacterium is agrobacterium A4, agrobacterium C58C1, agrobacterium ATCC15834, agrobacterium K599, agrobacterium LBA9402 and agrobacterium R1601.
When the agrobacterium A4 is placed in an LB liquid culture medium for activation, 50 mu g/mL kanamycin sulfate Kan needs to be added in the LB liquid culture medium; when the agrobacterium R1601 is placed in an LB liquid culture medium for activation, 50 mu g/mL kanamycin sulfate Kan needs to be added in the LB liquid culture medium; when the agrobacterium ATCC15834 is placed in an LB liquid culture medium for activation, 20 mu g/mL rifampicin Rif needs to be added into the LB liquid culture medium; when the agrobacterium K599 is placed in an LB liquid culture medium for activation, 50 mu g/mL streptomycin Strep needs to be added into the LB liquid culture medium.
(3) And (3) secondary activation of agrobacterium: centrifuging the agrobacterium liquid obtained in the step (2) for 10min at 5000 Xg, collecting thalli, resuspending and diluting the thalli by using an inducer to ensure that the obtained infected bacterial liquid OD600Activating for 10-40min with inducer at appropriate infection concentration of 0.4-1.0 to obtain infection solution;
the inducer is a liquid 1/2MS culture medium containing 100-500 mu mol/L acetosyringone, and the mass fraction of sucrose in the liquid 1/2MS culture medium is 5%;
preferably, the inducer in the step (3) is a liquid 1/2MS culture medium containing 300 mu mol/L acetosyringone, and the mass fraction of sucrose in the liquid 1/2MS culture medium is 5%.
(4) Infecting the explant with agrobacterium: treating an ammopiptanthus mongolicus explant, and infecting the treated ammopiptanthus mongolicus explant by using the infection liquid obtained in the step (3);
the time for infecting the explant is 10-30 min; the method for treating the ammopiptanthus mongolicus explant comprises the following steps: pricking a hole penetrating through the explant on the surface of the explant, pricking a hole not penetrating through the explant, scribing and cutting.
(5) Hairy root induction and propagation: putting the infected explants in an accelerant for co-culture for 4 d; hairy roots can be generated after being placed in the growth medium containing the antibiotics for about 15 days; as shown in fig. 2;
the promoter is a solid 1/2MS culture medium containing 100 mu mol/L-500 mu mol/L acetosyringone, and the mass fraction of sucrose in the solid 1/2MS culture medium is 5%.
Preferably, the promoter in the step (5) is solid 1/2MS culture medium containing 100 μmol/L acetosyringone, and the mass fraction of sucrose in the solid 1/2MS culture medium is 5%.
(6) And (3) amplification culture: cutting off from the explant when the hairy root of ammopiptanthus mongolicus is about 2cm long, putting the cut hairy root into a solid 1/2MS culture medium for in vitro rejuvenation and growth, and putting the hairy root into a liquid B5 culture medium for in vitro amplification culture when the hairy root is about 4cm long, as shown in figure 3. 2. Processing of experimental data and analysis of results
(1) Agrobacterium strain screening
The method selects six commonly used strains of agrobacterium A4, agrobacterium ATCC15834, agrobacterium C58C1, agrobacterium K599, agrobacterium LBA9402 and agrobacterium R1601 to respectively carry out infection induction on different explant parts of the Mongolian ammopiptanthus mongolicus at different infection concentrations.
The experimental result shows that the sensitivity of different agrobacterium strains for inducing hairy root tissues of mongolian ammopiptanthus mongolicus is as follows: agrobacterium A4>Agrobacterium C58C1>Agrobacterium ATCC15834>Agrobacterium K599, Agrobacterium R1601 and Agrobacterium LBA9402 could not be successfully induced. The A4 strain has the best induction effect, OD600At 0.6-0.7, the inductivity of the hairy root of the ammopiptanthus mongolicus can reach 68.2%, the multiplication coefficient of the hairy root is 7.74, and compared with other agrobacterium strains, the ammopiptanthus mongolicus has the advantages ofAnd (4) significance. Meanwhile, six different explant parts of the aseptic seedlings of the mongolian ammopiptanthus mongolicus, namely the epicotyl, the hypocotyl, the cotyledon, the true leaf, the stem and the root, are selected as receptor tissues. The experimental results show that the sensitivity of different mongolian ammopiptanthus mongolicus explant tissues is as follows: cotyledon>True leaf>The stem, epicotyl and root tissues are easy to brown and cannot be successfully induced. Specific results are shown in table 1.
TABLE 1 Agrobacterium strains and concentrations induced effects on hairy roots of ammopiptanthus mongolicus
The data in the table illustrate: (upper) inductivity and multiplication factor (lower)
The data in the table are followed by different lower case letters indicating significant differences at the 0.05 level
(2) Analysis of explant treatment mode on induction of hairy roots of ammopiptanthus mongolicus
Selecting ilex mongolica var mongolica cotyledons as explants, treating by adopting different treatment modes, including hole pricking (pricking and non-pricking), scribing, cutting and the like, and observing the inductivity of hairy roots. The experimental result shows that a certain number of small holes are drilled in the upper surface (paraxial surface) of the cotyledon explant without drilling, so that the induction of the hairy root is facilitated, the induction rate of the hairy root can be improved by more than 2 times, and the cotyledon explant has significance compared with other groups. Specifically, as shown in table 2.
TABLE 2 explant treatment Effect on hairy root Induction of Ammopiptanthus mongolicus
The data in the table are followed by different lower case letters indicating significant differences at the 0.05 level
(3) Effect of concentration of Accelerator and concentration and duration of inducer on hairy root
Before infection, an inducer containing acetosyringone with different concentrations is used for secondary activation of the agrobacterium strain for 30min, and an accelerant containing acetosyringone with different concentrations is used for co-culture, so that the induction rate and the proliferation coefficient of hairy roots of different groups are counted.
The experimental result shows that the induction rate and the proliferation coefficient of the inducer containing the acetosyringone on the hairy roots are both obviously improved, but the induction rate of the inducer containing the acetosyringone of 300 mu mol/L is not related to the concentration of the acetosyringone and can reach 63 percent to the maximum; under the condition of not using an inducer, the inducer and the multiplication coefficient of the acetosyringone-containing accelerant to the hairy root are not obviously changed, but after the inducer is used, the inducer and the multiplication coefficient of the hairy root can be obviously improved by using the accelerant. Wherein the inducer contains 300 mu mol/L acetosyringone, the accelerant contains 100 mu mol/L acetosyringone, and the obtained hairy root of the mongolian ammopiptanthus mongolicus has the highest induction rate and value-added coefficient. Specific results are shown in table 3.
TABLE 3 Effect of inducers and promoters containing different concentrations of acetosyringone on hairy roots of ammopiptanthus mongolicus
The data in the table illustrate: (upper) inductivity and multiplication factor (lower)
The data in the table are followed by different lower case letters indicating significant differences at the 0.05 level
According to the experimental results, liquid 1/2MS (sucrose mass fraction is 5%) containing 300 mu mol/L acetosyringone is selected as an inducer, and the growth condition of hairy roots of Mongolian ammopiptanthus mongolicus is observed under different induction times.
The experimental result shows that the induction rate of the inducer to the hairy roots is increased along with the increase of time within 0-30min, and the induction rate of the inducer to the hairy roots is reduced after 30 min. At 30min, the induction rate is the highest and is 68.15%, the proliferation coefficient is 7.74, and the induction rate is significantly different from other activation times. Specific results are shown in table 4.
TABLE 4 Induction Effect of Induction time of the Induction Agents on hairy roots of Ammopiptanthus mongolicus
The data in the table are followed by different lower case letters indicating significant differences at the 0.05 level
(4) Influence of Mongolian ammopiptanthus pubescens hairy root in vitro proliferation culture method on hairy root
In-vitro proliferation and growth of hairy roots of Mongolian ammopiptanthus mongolicus: a comparison was made between the solid and liquid culture of 1/2MS and B5 media which facilitate the growth of Ammopiptanthus mongolicus. When the hairy roots of the Mongolian ammopiptanthus mongolicus growing in vitro in a rejuvenation manner on an 1/2MS solid culture medium grow to about 4cm, the Mongolian ammopiptanthus mongolicus hairy roots are placed in different culture conditions for in vitro multiplication culture for 60 days, and the liquid culture mode is found to be very favorable for the multiplication of the biomass of the hairy roots of the Mongolian ammopiptanthus mongolicus, is more than 4.5 times that of the solid culture mode, and has significant difference; the B5 culture medium is more beneficial to the multiplication of hairy roots of ammopiptanthus mongolicus, is 1.55 times of 1/2MS culture medium, and has significant difference.
TABLE 5 Effect of culture on in vitro propagation of hairy roots of ammopiptanthus mongolicus
The data in the table are followed by different lower case letters indicating significant differences at the 0.05 level
(5) Analysis of formononetin content in hairy roots of ammopiptanthus mongolicus
Extracting hairy root of ammopiptanthus mongolicus with aerial medicinal part of wild adult plant (from Alxa, Hu and Duhui trees and stirrup region) by alcohol to obtain crude extract, and detecting content of formononetin as main medicinal component by HPLC analysis. The hairy roots obtained by the method can obviously improve the content of formononetin of the Mongolian ammopiptanthus mongolicus, and the content of 60d grown plants is obviously higher than that of field perennial plants.
TABLE 6 content of formononetin of ammopiptanthus mongolicus in hairy roots and wild plants
The data in the table are followed by different lower case letters indicating significant differences at the 0.05 level
In conclusion, the invention provides the induction and culture method of the hairy root system of the ammopiptanthus mongolicus, which can improve the yield of the formononetin, and the hairy root of the formononetin with high yield can be obtained by the method; the invention provides a novel inducer, which is a liquid 1/2MS culture medium containing 100 mu mol/L-500 mu mol/L acetosyringone, wherein the mass fraction of sucrose in the liquid 1/2MS culture medium is 5 percent, and the inducer is used for carrying out secondary activation on agrobacterium; compared with the traditional hairy root induction and culture method, the method adds the inducer secondary activation strain; according to the method, Mongolian ammopiptanthus mongolicus is infected by agrobacterium rhizogenes strains to induce hairy root tissues to grow, an in-vitro hairy root culture system is established, and the content of formononetin of Mongolian ammopiptanthus mongolicus is remarkably improved by utilizing the vigorous growth power of the hairy root tissues and the efficient biosynthesis capacity of the hairy root tissues on secondary metabolites and is improved by 14 times compared with that of wild plants; the hairy roots of the Mongolian ammopiptanthus mongolicus have the advantages of quick growth, short period, no limitation of seasons, high relative yield of secondary metabolites and the like, and can be used for industrially producing the formononetin of the Mongolian ammopiptanthus mongolicus. Provides an available way for the industrial production of the secondary metabolite of the mongolian ammopiptanthus mongolicus by utilizing hairy roots in the future, and is beneficial to the protection and sustainable utilization of the endangered resource plants on the other hand.
Claims (10)
1. An induction and culture method of hairy root system of ammopiptanthus mongolicus for improving formononetin yield is characterized by comprising the following steps:
(1) obtaining an ammopiptanthus mongolicus explant: selecting mongolian ammopiptanthus mongolicus seeds, sterilizing, and carrying out aseptic culture to obtain aseptic seedlings and obtain explants;
(2) agrobacterium selection and activation: selecting agrobacterium strains, selecting monoclonals from a culture plate, placing the monoclonals in an LB liquid culture medium containing corresponding antibiotics, and performing shaking culture; the bacterial liquid is absorbed and inoculated in an LB liquid culture medium for amplification culture until the bacterial liquid OD600Is 1.5;
(3) and (3) secondary activation of agrobacterium: centrifuging the agrobacterium liquid obtained in the step (2), collecting thalli, resuspending and diluting the thalli by using an inducer to ensure that the bacterial liquid OD is obtained600Standing the activated bacterium liquid for 10-40min to obtain an infection liquid, wherein the bacterial liquid is 0.4-1.0;
(4) infecting the explant with agrobacterium: treating the ammopiptanthus mongolicus explant, and infecting the ammopiptanthus mongolicus explant by using the infection liquid obtained in the step (3);
(5) hairy root induction and propagation: co-culturing the infected explant in promoter; then the culture medium is placed in a growth medium containing corresponding antibiotics and can be sent out by hairy roots;
(6) and (3) amplification culture: cutting off from the explant when the hairy root of ammopiptanthus mongolicus grows to 1-3cm, putting the cut hairy root into a solid 1/2MS culture medium for in vitro rejuvenation and growth, and putting the hairy root into a liquid B5 culture medium for in vitro amplification culture when the hairy root grows to 4-5 cm;
the inducer in the step (3) is a liquid 1/2MS culture medium containing 100-500 mu mol/L acetosyringone, and the mass fraction of sucrose in the liquid 1/2MS culture medium is 5%.
2. The method for inducing and culturing hairy root system of Mongolian ammopiptanthus capable of improving formononetin production as claimed in claim 1, wherein the inducing agent in step (3) is liquid 1/2MS culture medium containing 300 μmol/L acetosyringone, and the mass fraction of sucrose in the liquid 1/2MS culture medium is 5%.
3. The method for inducing and culturing hairy root system of Mongolian ammopiptanthus capable of improving formononetin production as claimed in claim 1, wherein the promoter in step (5) is solid 1/2MS culture medium containing 100 μmol/L-500 μmol/L acetosyringone, and the mass fraction of sucrose in the solid 1/2MS culture medium is 5%.
4. The method for inducing and culturing hairy root system of Mongolian ammopiptanthus capable of improving formononetin production as claimed in claim 3, wherein the promoter in step (5) is solid 1/2MS culture medium containing 100 μmol/L acetosyringone, and the mass fraction of sucrose in the solid 1/2MS culture medium is 5%.
5. The method for inducing and culturing hairy root line of ammopiptanthus mongolicus with enhanced formononetin production as claimed in claim 1, wherein the ammopiptanthus mongolicus explant in the step (1) comprises epicotyl, hypocotyl, cotyledon, true leaf, stem and root.
6. The method for inducing and culturing hairy root line of ammopiptanthus mongolicus for improving formononetin yield of claim 1, wherein the time for infecting the explant in the step (4) is 10-30 min.
7. The method for inducing and culturing hairy root line of ammopiptanthus mongolicus for improving formononetin yield of claim 1, wherein the method for treating ammopiptanthus mongolicus explants in step (4) comprises: pricking holes on the surface of the explant, scribing and cutting.
8. The method for inducing and culturing hairy root system of ammopiptanthus mongolicus for improving formononetin yield of claim 7, wherein the holes are drilled on the surface of the explant and do not penetrate the explant.
9. The method for inducing and culturing hairy root line of Mongolian ammopiptanthus capable of improving formononetin production as claimed in claim 1, wherein the Agrobacterium of step (2) is any one of Agrobacterium A4, Agrobacterium C58C1, Agrobacterium ATCC15834, Agrobacterium K599, Agrobacterium LBA9402 and Agrobacterium R1601.
10. Use of the method of induction and culture of hairy root system of ammopiptanthus mongolicus for increasing formononetin yield as claimed in any one of claims 1 to 9 for preparing ammopiptanthus mongolicus.
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