CN118266404A - Induction method for rapidly obtaining hairy roots of sweet clover - Google Patents
Induction method for rapidly obtaining hairy roots of sweet clover Download PDFInfo
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Abstract
The invention discloses an induction method for rapidly obtaining hairy roots of sweet clover, which comprises the following steps: s1, preparing sweet clover aseptic seedlings, and obtaining sweet clover aseptic seedlings with two cotyledons spread; s2, transforming agrobacterium rhizogenes with a vector carrying GFP genes, coating and inoculating the transformed agrobacterium rhizogenes onto an LB solid culture medium, activating, selecting single bacterial colonies, inoculating the single bacterial colonies into an LB liquid culture medium, adding kanamycin and rifampicin, carrying out shake culture until logarithmic phase, centrifugally collecting thalli, re-suspending the thalli by using an MS liquid culture medium, adding acetosyringone, and carrying out shake culture for 30 min-2 hours to prepare bacterial suspension; s3, cutting roots of the sweet clover sterile seedlings with the cotyledons unfolded, soaking and infecting the sweet clover sterile seedlings in agrobacterium rhizogenes bacterial suspension for 3-5 min hours, taking out seedlings, placing the infected sweet clover seedlings on a co-culture medium for co-culture for 2d in a dark treatment mode, taking out sweet clover seedlings, and placing the sweet clover seedlings on an induction medium to induce hairy roots. A large number of hairy roots are easily obtained by the present invention.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an induction method for rapidly obtaining hairy roots of sweet clover.
Background
The sweet clover (Melilotus officinalis (L.) pall.) is also called yellow flower grass, sweet clover, sweet Ma Liaomu sweet clover and sweet spot, is a leguminous annual or biennial herb plant, has strong environmental adaptability, strong stress resistance, is moist-loving, drought-resistant, cold-resistant and barren-resistant, has low requirement on soil, and can be planted in saline-alkali areas where many pastures are difficult to grow. After being planted in the light saline-alkali soil, the soil saline-alkali can be reduced, the soil fertility can be improved, and the method is one of main selected plants for ecological restoration and reconstruction of salinized abandoned farmland. The sweet clover has wide distribution, strong adaptability and higher nutritional value. Can be used for green feeding, silage and sun-curing hay to prepare grass meal, which is not only a good forage grass, but also a good protein feed. Sweet clover contains a variety of mineral nutrients and trace elements, which are very important for increasing the nutrition and soil fertility of livestock. The coumarin content is relatively low, so that the coumarin is a good germplasm resource.
At present, research on sweet clover mainly focuses on cultivation technology research, chemical component identification, molecular marker development, genetic diversity evaluation of sweet clover plants and the like, but research on mutants and molecular mechanisms of sweet clover is less, and at present, methods for carrying out gene transformation on sweet clover by using agrobacterium rhizogenes exist, but the methods have more steps, complex operation and poor transformation efficiency, so that deep analysis on gene functions of sweet clover is limited.
Disclosure of Invention
The invention aims to provide an induction method for quickly obtaining hairy roots of sweet clover, which can quickly induce sweet clover to generate hairy roots by using agrobacterium rhizogenes.
In order to solve the problems in the prior art, the technical scheme adopted by the invention is as follows:
An induction method for rapidly obtaining hairy roots of sweet clover comprises the following steps:
s1, preparing sweet clover aseptic seedlings, and obtaining sweet clover aseptic seedlings with two cotyledons spread;
S2, preparing agrobacterium rhizogenes:
Transforming agrobacterium rhizogenes by using a carrier carrying GFP genes, coating and inoculating the transformed agrobacterium rhizogenes to an LB solid culture medium containing antibiotics, activating, picking single bacterial colonies, inoculating to a LB liquid culture medium containing antibiotics, shake-culturing to logarithmic phase, centrifugally collecting thalli, re-suspending by using an MS liquid culture medium, adding acetosyringone, shake-culturing for 30 min-2 hours to prepare bacterial suspension;
S3, inducing and sterilizing hairy roots: cutting the sweet clover aseptic seedlings obtained in the step S1 into roots, soaking and infecting the sweet clover aseptic seedlings in agrobacterium rhizogenes bacterial suspension for 3-5 min hours, taking out the seedlings, placing the infected sweet clover seedlings on a co-culture medium for co-culture for 2 d in a dark process, taking out the sweet clover seedlings, and placing the sweet clover seedlings on an induction medium to induce hairy roots.
Further, the method also comprises the step S4 of detecting hairy roots:
And (3) PCR detection: extracting the hairy root total DNA as a detection template, taking agrobacterium rhizogenes plasmid DNA as a positive control, detecting a target gene which is GFP, and detecting a product after PCR amplification by using 1% agarose gel electrophoresis;
fluorescence detection: fluorescence detection was performed on the roots after induction using a fluorescence exciter.
Preferably, the agrobacterium rhizogenes is K599 or LBA9402.
Further, the method for transforming agrobacterium rhizogenes with a vector carrying GFP gene comprises the steps of: thawing Agrobacterium rhizogenes competent cells on ice, adding 3 mu L of plasmid carrying GFP gene after thawing on ice, standing on ice for 30min, freezing 1 min in liquid nitrogen, adding 500 mu L of LB liquid medium after water bath 3 min at 37 ℃, shaking and culturing 3h at 200 ℃ and rpm, centrifuging and concentrating bacterial liquid, and reserving 50 mu L of LB liquid medium for re-dissolving thalli to obtain the transformed Agrobacterium rhizogenes.
Further, the preparation method of the aseptic seedling of sweet clover in the step S1 comprises the following steps of
Sweet clover seeds with full seeds are selected, the sweet clover seeds are sterilized by shaking with ethanol with the volume concentration of 75 percent for 30-60 s, sterilized with NaClO solution with the volume concentration of 3 percent for 8-10 min, washed with sterile water for 3-5 times, inoculated on a 1/2 MS solid culture medium, and cultured until two cotyledons are unfolded under the conditions that the temperature is 23-25 ℃, the illumination intensity is 15000 lux and the illumination time is 16 h/d.
Preferably, the 1/2MS solid medium in the step S1 comprises the following components: 2.21 g/L MS minimal medium +30 g/L sucrose +7.8 g/L agar, pH 5.8-6.0.
Preferably, the concentration of the bacterial suspension in the step S2 is: the OD 600 value of the bacterial suspension is 0.4-1.0.
Preferably, the composition of the solid medium containing the antibiotic LB in the step S2 is as follows: 25 g/L LB broth+16 g/L agar+50 mg/L kanamycin+25 mg/L rifampicin; the LB liquid culture medium containing the antibiotics comprises the following components: 25 g/L LB broth+50 mg/L kanamycin+25 mg/L rifampin.
Preferably, the co-culture medium in the step S3 has the following composition: 2.21 g/L MS minimal medium+30 g/L sucrose+7.8 g/L agar+100. Mu.M/L acetosyringone; the conditions for co-cultivation were: the temperature is 23-25deg.C, and the darkness is achieved.
Preferably, the induction medium in the step S3 comprises the following components: 2.21 g/L MS minimal medium +30 g/L sucrose +7.8 g/L agar +400 mg/L timentin, pH 5.8-6.0; the conditions of the induction culture are as follows: the temperature is 23-25 ℃, and the illumination time is 16 h/d.
The beneficial effects obtained by the invention include the following aspects:
1) The induction method for rapidly obtaining the hairy roots of sweet clover does not need to perform preculture of explants, simplifies experimental steps, is easy to operate due to fewer operation steps, and greatly reduces the possibility of pollution.
2) The invention adopts a method of soaking infection after root cutting, the operation method is simple, and the T-DNA region gene group on the Ri plasmid of the agrobacterium rhizogenes is easier to transfer and integrate into the explant cells; the induction culture medium can induce hairy roots to generate and effectively inhibit the growth of agrobacterium rhizogenes and other bacteria; the conversion rate of agrobacterium rhizogenes is improved.
3) The invention establishes a high-efficiency induction system of hairy roots of sweet clover, and the hairy roots obtained by the invention have short period and large quantity of hairy roots, and the induction rate of the hairy roots is 90-100%.
Drawings
FIG. 1 is a schematic diagram of the hairy root induction system of sweet clover;
FIG. 2 shows the induction of hairy roots of trifolith by varying concentrations and times of Agrobacterium K599 as described in example 2; in the figure:
FIG. 2A shows the induction of hairy roots of sweet clover by K599 Agrobacterium OD 600 at 0.4 for 3 minutes;
FIG. 2B shows the induction of hairy roots of sweet clover by Agrobacterium tumefaciens OD 600 at 0.6 for 3 minutes K599;
FIG. 2C shows the induction of hairy roots of sweet clover by K599 Agrobacterium OD 600 at 0.8 for 3 minutes;
FIG. 2D shows the induction of hairy roots of sweet clover by Agrobacterium tumefaciens OD 600 at a value of 1.0 for 3 minutes;
FIG. 2E shows the induction of hairy roots of sweet clover by K599 Agrobacterium OD 600 at 0.4 for 5 minutes;
FIG. 2F K599 Agrobacterium OD 600 at 0.6 for 5 minutes for the induction of hairy roots of sweet clover;
FIG. 2G is a graph showing the induction of hairy roots of sweet clover by K599 Agrobacterium OD 600 at 0.8 for 5 minutes;
FIG. 2H shows the induction of hairy roots of sweet clover by K599 Agrobacterium OD 600 at 1.0 for 5 minutes;
FIG. 3 shows the induction of trifolith hairy roots at various concentrations and times of LBA9402 Agrobacterium as described in example 1; in the figure:
FIG. 3A shows induction of hairy roots of sweet clover by LBA9402 Agrobacterium OD 600 at 0.4 for 3 minutes;
FIG. 3B shows induction of hairy roots of sweet clover by LBA9402 Agrobacterium OD 600 at 0.6 for 3 minutes;
FIG. 3C shows induction of hairy roots of sweet clover by LBA9402 Agrobacterium OD 600 at 0.8 for 3 minutes;
FIG. 3D shows induction of hairy roots of sweet clover by LBA9402 Agrobacterium OD 600 at 1.0 for 3 minutes;
FIG. 3E shows induction of hairy roots of sweet clover by LBA9402 Agrobacterium OD 600 at 0.4 for 5 minutes;
FIG. 3F shows induction of hairy roots of sweet clover by LBA9402 Agrobacterium OD 600 at 0.6 for 5 minutes;
FIG. 3G shows induction of hairy roots of sweet clover by LBA9402 Agrobacterium OD 600 at 0.8 for 5 minutes;
FIG. 3H shows induction of hairy roots of sweet clover by LBA9402 Agrobacterium OD 600 at 1.0 for 5 minutes;
FIG. 4 shows the expression of GFP from hairy roots of sweet clover;
FIG. 5 shows the PCR detection of the green root GFP gene of sweet clover.
Detailed Description
The technical solutions of the embodiments of the present application will be clearly described below with reference to the drawings in the embodiments of the present application, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments. All other embodiments, which are obtained by a person skilled in the art based on the embodiments of the present application, fall within the scope of protection of the present application.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The components of the culture medium used in the application are as follows:
The solid culture medium containing the antibiotics LB comprises the following components: 25 g/L LB broth+16 g/L agar+50 mg/L kanamycin+25 mg/L rifampicin;
the components of the LB liquid medium containing the antibiotics are as follows: 25 g/L LB broth+50 mg/L kanamycin+25 mg/L rifampin;
The LB liquid medium comprises the following components: 25 g/L LB broth;
The co-culture medium comprises the following components: 2.21 g/L MS minimal medium +30 g/L sucrose +7.8 g/L agar +100. Mu.M/L acetosyringone, pH 5.8-6.0;
the induction medium comprises the following components: 2.21 g/L MS minimal medium +30 g/L sucrose +7.8 g/L agar +400 mg/L timentin, pH 5.8-6.0;
The 1/2MS solid culture medium comprises the following components: 2.21 g/L MS minimal medium +30 g/L sucrose +7.8 g/L agar, pH 5.8-6.0;
The MS liquid culture medium comprises the following components: 4.43 The g/L MS minimal medium is added with 30 g/L sucrose, and the pH is 5.8-6.0.
Embodiment 1, as shown in fig. 1, provides an induction method for rapidly obtaining hairy roots of sweet clover, which specifically comprises the following steps:
s1, obtaining sweet clover aseptic seedlings:
Selecting 100 seeds of sweet clover seeds with full seeds, vibrating and sterilizing 40 s in an ultra-clean workbench by using 2 mL volumes of 75% ethanol, sterilizing 9 min by using 2 mL volumes of 3% NaClO solution, cleaning with a proper amount of sterile water for 4 times, sucking residual water on the surfaces of the seeds by using sterile filter paper, inoculating the seeds on a 1/2 MS solid culture medium, and culturing at the temperature of 24 ℃ under the condition of the illumination intensity of 15000 lux and the illumination time of 16 h/d until two cotyledons are stretched.
S2, activating agrobacterium rhizogenes and preparing agrobacterium rhizogenes bacterial suspension:
Thawing Agrobacterium rhizogenes LBA9402 competent cells on ice, adding 3 mu L of plasmid carrying GFP genes into 50 mu L of competent cells after thawing on ice, standing on ice for 30min, freezing 1 min in liquid nitrogen, adding 500 mu L of LB liquid medium after cooling 3 min in water bath at 37 ℃,28 ℃,200 rpm shake culturing 3 h, centrifuging concentrated bacterial liquid, reserving 50 mu L of LB liquid medium for re-dissolving bacterial cells, coating and inoculating the re-melted bacterial liquid onto LB solid medium containing antibiotics, activating at 28 ℃, picking single colony to inoculate into LB liquid medium containing antibiotics 50mL, shake culturing at 28 ℃ for logarithmic growth phase under 200 r/min, centrifuging 15min to collect bacterial cells, resuspending with 100 mu L of 100 mM/mL acetosyringone, shake culturing at 28 ℃ under 200 r/min for 30 min-2h to prepare bacterial suspension, wherein the bacterial suspension concentration is: the OD600 of the bacterial suspension was 0.6.
S3, inducing hairy roots:
Cutting the aseptic seedling of sweet clover with cotyledons spread in an ultra-clean workbench, cutting off the aseptic seedling at the hypocotyl by a surgical knife, then placing the aseptic seedling in agrobacterium rhizogenes bacterial suspension for soaking and infection, immersing the hypocotyl incision of the sweet clover in the bacterial suspension, taking out the seedling after soaking for 3 min, sucking the superfluous bacterial liquid on the surface by using sterile filter paper, inoculating the aseptic seedling to a dark place on a co-culture medium for co-culture for 2d, and controlling the temperature to 24 ℃; the co-culture medium is a solid culture medium, the height is 1/3-1/2 of the height of the plate, then young sweet clover is taken out and inoculated on the induction culture medium to induce hairy roots, 10 young sweet clover seedlings are inoculated on each induction culture medium, the culture medium is placed at 24 ℃, the illumination intensity is 15000 lux, the illumination time is 16 h/d for sterilization culture, and the expansion of the root cutting position of the young sweet clover seedlings can be seen after 3 d; 5-7 d it was observed that hairy roots were grown at the cut of most seedlings. As shown in FIG. 4, after 14 d, the hairy roots grow to 2-4 cm, and green fluorescence is observed by detecting the hairy roots with a fluorescence exciter. GFP genes were detected in hairy roots by PCR detection, as shown in FIG. 5.
The incidence and conversion of hairy roots of example 1 were observed and calculated:
The calculation method of the occurrence rate of the hairy roots comprises the following steps: hairy root occurrence= (hairy root number of occurrences/total number of occurrences) ×100%.
Number of hairy roots = total number of hairy roots/total number of hairy roots plants.
The calculation results are that: incidence of hairy roots in this example: 100%; number of hairy roots: 4.73.
In example 2, this example provides a method for inducing the rapid onset of hairy roots of sweet clover, which differs from example 1 only in that Agrobacterium rhizogenes in step S2 is K599, and the remainder is the same as in example 1.
The calculation results are that: incidence of hairy roots in this example: 93.75%; number of hairy roots: 3.3.
Example 3 effect of different strains on the induction rate of hairy roots of sweet clover:
The effect of the two Agrobacterium rhizogenes bacteria solutions on the induction rate of hairy roots of sweet clover was compared between K599 and LBA9402, and the results are shown in FIGS. 2 and 3.
As can be seen by comparing FIGS. 2 and 3, the induction rate of Agrobacterium rhizogenes LBA9402 to hairy roots of Melilotus is significantly greater than that of Agrobacterium rhizogenes K599.
Example 4 influence of the time of infection soaking on the induction rate of hairy roots of sweet clover
This example investigated the effect of different infection soak times on the infection outcome (other conditions except for variables were identical to examples 1, 2):
According to the methods described in example 1 and example 2, the soaking and infecting time of sweet clover in the step S3 is set to 3min and 5min respectively, and the results of the influence of different soaking and infecting times on the induction rate of the hairy roots of sweet clover are shown in fig. 2 and 3, and the results show that the induction rate of the hairy roots of sweet clover at the 3min infection time is greater than the induction rate of 5min both by agrobacterium rhizogenes K599 and LBA 9402.
Example 5 Effect of bacterial suspension concentration on the rate of induction of hairy roots in sweet clover
In this example, the influence of different bacterial liquid OD 600 values on the infection result was studied (except for the variables, the other conditions were the same as in example 1 and example 2 respectively):
According to the methods described in examples 1 and 2, the agrobacterium rhizogenes was prepared into bacterial suspensions with OD 600 value of 0.4, 0.6, 0.8 and 1.0, and the results of the test of the effect of different bacterial suspension concentrations on the hairy root induction rate of melilotus are shown in fig. 2 and 3, which show that, in a certain range, the hairy root induction rate of melilotus is increased and decreased with the increase of the agrobacterium rhizogenes bacterial suspension OD600 value, when the agrobacterium bacterial suspension OD 600 value is 0.6, the hairy root induction rate is the highest, and when the bacterial suspension OD600 value exceeds 0.6, the hairy root induction rate is decreased again, and therefore, the hairy root bacterial suspension OD 600 value is about 0.6 and is a suitable concentration of bacterial suspension. When the concentration of the bacterial suspension is too low, the quantity of agrobacterium rhizogenes is small, the contact probability of thalli and aseptic seedlings is small, and the infection efficiency is affected; when the concentration is too high, the excessive bacteria can cause damage to aseptic seedling cells, which is not only unfavorable for the combination of the bacteria and aseptic seedlings, but also can increase the difficulty of the subsequent sterilization operation.
Example 6 detection of hairy roots:
And (3) PCR detection: the total DNA of trifoliter hairy roots obtained in the step S3 of the example 1 and the example 2 is respectively extracted by adopting a CTAB method and used as a detection template, the converted agrobacterium rhizogenes plasmid DNA is used as a positive control, the target gene GFP is detected, and the products after PCR amplification are detected by using 1% agarose gel electrophoresis. The gene amplification primers synthesized by Savin Innovation were 5 'AAGTTCACCTTGATGCCGTTCT 3' (SEQ ID NO. 1) and 5 'AGTGCTTCAGCCGCTACCC 3' (SEQ ID NO. 2); the PCR amplification conditions were: pre-denaturation at 95 ℃ 3 min; denaturation at 95℃for 15 s, annealing at 50℃for 15 s, elongation at 72℃for 15 s,35 cycles; finally, the extension is carried out at 72 ℃ for 5 min. The detection shows that the obtained hairy root genome of the sweet clover contains GFP genes carried by a positive control plasmid, which indicates that the hairy root of the sweet clover is integrated with the T-DNA fragment of the agrobacterium rhizogenes plasmid.
Fluorescence detection: the hairy root was fluorescence detected using a fluorescence exciter.
As shown in FIG. 4, after 14 d, the hairy roots were grown to 2-4 cm, and green fluorescence was observed in the hairy roots obtained in example 1 and example 2 by detecting the hairy roots with a fluorescence exciter.
Comparative example 1, which provides an induction method for rapidly obtaining hairy roots of sweet clover, is different from example 1 only in that the induction medium used in step S3 has the following composition:
30.12 g/L B5 g/L sucrose+7. g/L agar+400 mg/L timentin, pH 5.8-6.0, the other procedures were the same as in example 1.
Incidence of hairy roots in this comparative example: 78.57%; the number of hairy roots was 2.03.
The test results show that the occurrence rate of sweet clover root is reduced due to the difference of basal culture mediums in the induction culture mediums.
Comparative example 2, which provides an induction method for rapidly obtaining hairy roots of sweet clover, differs from example 1 only in that the co-culture medium in step S3 has the following composition: 2.21 g/L MS minimal medium +30 g/L sucrose +7.8 g/L agar, pH 5.8-6.0; the other operation steps were the same as in example 1.
Incidence of hairy roots in this comparative example: 82.86% of the total weight of the product; the number of hairy roots was 2.78.
The test result shows that the occurrence rate of the root of sweet clover is reduced because acetosyringone is not added into the symbiotic culture medium.
The embodiments of the present application have been described above with reference to the accompanying drawings, but the present application is not limited to the above-described embodiments, which are merely illustrative, not restrictive, and many forms may be made by those having ordinary skill in the art without departing from the spirit of the present application and the scope of the claims, which are to be protected by the present application.
Claims (10)
1. An induction method for rapidly obtaining hairy roots of sweet clover is characterized by comprising the following steps:
S1, preparing sweet clover aseptic seedlings with two unfolded cotyledons;
S2, preparing agrobacterium rhizogenes:
Transforming agrobacterium rhizogenes by using a carrier carrying GFP genes, coating and inoculating the transformed agrobacterium rhizogenes to an LB solid culture medium containing antibiotics, activating, picking single bacterial colonies, inoculating to a LB liquid culture medium containing antibiotics, shake-culturing to logarithmic phase, centrifugally collecting thalli, re-suspending by using an MS liquid culture medium, adding acetosyringone, shake-culturing for 30 min-2 hours to prepare bacterial suspension;
s3, inducing and sterilizing hairy roots: cutting the sweet clover aseptic seedlings obtained in the step S1 into roots, soaking and infecting the sweet clover aseptic seedlings in agrobacterium rhizogenes bacterial suspension for 3-5 min hours, taking out the seedlings, placing the infected sweet clover seedlings on a co-culture medium for co-culture in a dark treatment manner, taking out the sweet clover seedlings, and placing the sweet clover seedlings on an induction medium to induce hairy roots.
2. The method for inducing sweet clover hairy roots rapidly according to claim 1, further comprising the step of S4 hairy root detection:
And (3) PCR detection: extracting the hairy root total DNA as a detection template, taking agrobacterium rhizogenes plasmid DNA as a positive control, detecting a target gene which is GFP, and detecting a product after PCR amplification by using 1% agarose gel electrophoresis;
fluorescence detection: fluorescence detection was performed on the roots after induction using a fluorescence exciter.
3. An induction method for rapidly obtaining hairy roots of sweet clover according to claim 1 or2, characterized in that: the agrobacterium rhizogenes is K599 or LBA9402.
4. A method of inducing rapid onset of hairy roots of sweet clover according to claim 3, wherein:
The method for transforming the vector carrying the GFP gene into agrobacterium rhizogenes comprises the following steps: thawing Agrobacterium rhizogenes competent cells on ice, adding plasmid carrying GFP gene after thawing on ice, standing on ice, freezing in liquid nitrogen, adding LB liquid culture medium after water bath, shake culturing, centrifuging, concentrating bacterial liquid, retaining part of LB liquid culture medium, and dissolving thallus to obtain transformed Agrobacterium rhizogenes.
5. The method for inducing sweet clover hairy roots to be obtained rapidly according to claim 1 or 2, wherein the preparation method of sweet clover aseptic seedlings in step S1 comprises the following steps:
Sweet clover seeds with full seeds are selected, the sweet clover seeds are sterilized by shaking with ethanol with the volume concentration of 75% for 30-60 s, sterilized with NaClO solution with the volume concentration of 3% for 8-10 min, washed with sterile water for 3-5 times, inoculated on a 1/2 MS solid culture medium, and cultured until two cotyledons are unfolded under the conditions that the temperature is 23-25 ℃, the illumination intensity is 15000 lux and the illumination time is 16 h/d.
6. The method for inducing sweet clover hairy roots according to claim 5, wherein the 1/2 MS solid medium in step S1 comprises the following components: 2.21 g/L MS minimal medium +30 g/L sucrose +7.8 g/L agar, pH 5.8-6.0.
7. The method for inducing sweet clover hairy roots rapidly according to claim 1 or 2, wherein the OD 600 of the bacterial suspension in step S2 is 0.4-1.0.
8. The method for inducing sweet clover hairy roots rapidly according to claim 1 or 2, wherein the composition of the solid medium containing antibiotic LB in step S2 is: 25 g/L LB broth+16 g/L agar+50 mg/L kanamycin+25 mg/L rifampicin; the LB liquid culture medium containing the antibiotics comprises the following components: 25 g/L LB broth+50 mg/L kanamycin+25 mg/L rifampin.
9. The method for rapidly obtaining hairy roots of sweet clover according to claim 1, wherein the co-culture medium in the step S3 comprises the following components: 2.21 g/L MS minimal medium+30 g/L sucrose+7.8 g/L agar+100. Mu.M/L acetosyringone; the conditions for co-cultivation were: the temperature is 23-25deg.C, and the darkness is achieved.
10. The method for rapidly obtaining hairy roots of sweet clover according to claim 1, wherein the induction medium in the step S3 comprises the following components: 2.21 g/L MS minimal medium +30 g/L sucrose +7.8 g/L agar +400 mg/L timentin, pH 5.8-6.0; the conditions of the induction culture are as follows: the temperature is 23-25 ℃, and the illumination time is 16 h/d.
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