CN113652446A

CN113652446A - Agrobacterium rhizogenes-mediated one-step transformation method for hairy roots of caragana intermedia

Info

Publication number: CN113652446A
Application number: CN202111103769.2A
Authority: CN
Inventors: 万永青; 杨闯; 柳金华; 李国婧; 王瑞刚
Original assignee: Inner Mongolia Agricultural University
Current assignee: Inner Mongolia Agricultural University
Priority date: 2021-09-22
Filing date: 2021-09-22
Publication date: 2021-11-16
Anticipated expiration: 2041-09-22
Also published as: CN113652446B

Abstract

The invention discloses a one-step transformation method of hairy roots of caragana intermedia mediated by agrobacterium rhizogenes, which comprises the following steps: s1, electrically transforming a plasmid containing an e-GFP expression vector into an agrobacterium rhizogenes K599 competent cell; s2. preparation of OD_600nm0.8-0.95 agrobacterium rhizogenes suspension, and adding acetosyringone; s3, culturing the bacterial paste used for infection; s4, selecting caragana intermedia seedlings, beveling 1cm below cotyledonary nodes of the seedlings, discarding the lower parts, infecting the cut, transplanting the seedlings back to the soil, watering the seedlings with 1/4B5 culture medium, and placing the seedlings in the light for cultureCulturing in a culture box. Compared with a tissue culture method for transforming the hairy root of the intermediate caragana, the method has the advantages of simpler operation, no need of preparing aseptic seedlings, shorter detection time, high gene expression level and convenience in detection.

Description

Agrobacterium rhizogenes-mediated one-step transformation method for hairy roots of caragana intermedia

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to a one-step transformation method of hairy roots of caragana intermedia mediated by agrobacterium rhizogenes.

Background

The concept of hairy roots was first proposed by STEWART in 1900. In 1907, Smith and Townsend found that Agrobacterium rhizogenes could infect plants to produce hairy roots. The agrobacterium plasmid mediated process is one efficient plant gene engineering transforming process. Most of the transgenic plants were obtained by mediating the Agrobacterium tumefaciens Ti plasmid. Chilton reported in 1982 that hairy roots were caused by the Ri plasmid of Agrobacterium rhizogenes. So far, the hairy root technology has entered a completely new era. Related studies of Ri-plasmid mediated genetic transformation of plant hairy roots have been developed across numerous plant species. The hairy root culture technology is mainly used for synthesizing plant secondary metabolites and promoting gene expression, and also serves as an important technical means for enhancing plant adaptability and improving plant stress tolerance. The study of plant hairy root culture started in 1998. Panyan et al in 2015 discussed the technique for inducing hairy root of Panax notoginseng and its application. And in 2018, Maoya Fangqi and the like establish a cotton hairy root transformation system. In 2020 Roughani et al published the research progress of transgenic Polygonum multiflorum hairy root. At present, hairy root induction has been widely applied to herbaceous plants, woody plants, shrubs and the like, and mainly used for economic plants and medicinal plants. In recent years, the established plant hairy root transformation system comprises corn, spinach, liquorice, broccoli, black nightshade, stevia rebaudiana, cucumber, salvia miltiorrhiza and the like.

Caragana intermedia (Caragana intermedia) is shrub of Caragana in Caragana of Caragana, and is a perennial dry-grown deciduous shrub species in loess plateau areas. The caragana plants are widely distributed in inner Mongolia areas and are used as dominant species for preventing wind, fixing sand and keeping water and soil in dry areas of Ningxia, Gansu, Shanxi and Shanxi. The adaptability and the stress resistance of the middle caragana can be improved through the induction of the hairy roots, the variety is improved, and the method has important significance for production, life and environmental construction. At present, a genetic transformation system of the hairy root of the middle caragana is not established. A method for transforming the hairy root of caragana intermedia by a one-step method is also rarely reported. Therefore, it is necessary to provide a one-step transformation method of caragana intermedia hairy roots mediated by agrobacterium rhizogenes, which provides a convenient way for researching caragana microphylla gene functions and lays a foundation for further research of caragana microphylla genetic transformation.

Disclosure of Invention

In order to overcome the defects and shortcomings in the prior art, the invention aims to provide an agrobacterium rhizogenes-mediated one-step transformation method for the hairy roots of caragana intermedia.

An agrobacterium rhizogenes-mediated one-step transformation method for middle caragana hairy roots, comprising the following steps:

s1, electrically transforming a plasmid containing an e-GFP expression vector into an agrobacterium rhizogenes K599 competent cell;

s2, inoculating the agrobacterium rhizogenes K599 obtained in the step S1 to an LB liquid culture medium for culture, and preparing OD_600nm0.8-0.95 of agrobacterium rhizogenes suspension, and adding acetosyringone with the final concentration of 25-100 mu mol/mL into the prepared agrobacterium rhizogenes suspension;

s3, centrifuging a part of the agrobacterium rhizogenes suspension added with the acetosyringone in the step S2, removing supernatant, uniformly mixing the rest precipitate, absorbing the uniformly mixed precipitate, adding the uniformly mixed precipitate into an LB solid culture medium for coating, putting the coated precipitate into an incubator for culturing bacterial ointment for infection, and performing the operations at room temperature of an ultra-clean workbench;

s4, selecting a middle caragana seedling, beveling 1cm below a cotyledon node of the middle caragana seedling, discarding the lower part, soaking a cut of the cotyledon node of the middle caragana seedling in a dark environment, scraping the bacterial paste obtained in the step S3 after the bacterial paste is scraped, directly transplanting the middle caragana seedling back to the soil, pouring 1/4B5 culture medium on the middle caragana seedling, placing the middle caragana seedling in an illumination incubator for culture, and opening an air vent of a seedling tray of the middle caragana seedling after 12-20 days.

Preferably, the e-GFP reporter gene-containing expression vector is pEGAD.

Preferably, the OD of the Agrobacterium rhizogenes suspension described in step S2_600nmThe value was 0.9 and the final acetosyringone concentration was 50. mu. mol/mL.

Preferably, the culture conditions of the bacterial paste used for the infection in step S3 are: the temperature of the incubator is set to 37 ℃, and the culture time is 10-16 hours.

Preferably, in the step S4, the soaking time at the cut of the cotyledon node of the middle caragana seedling is 1min, and the thickness of the bacterial paste at the cut is 0.1-0.5 mm.

Preferably, the 1/4B5 medium is used in an amount of 5mL in step S4.

Preferably, the step S2 specifically includes the following steps: inoculating the Agrobacterium rhizogenes K599 obtained in the step S1 to LB liquid medium, and culturing at 28 ℃ with shaking to OD_600nmObtaining a primary bacterial suspension for 0.9, then transferring the primary bacterial suspension into a fresh LB liquid culture medium again, and carrying out shake culture on the primary bacterial suspension to OD at 28 DEG C_600nmAfter the concentration is 0.8-0.95, obtaining agrobacterium rhizogenes bacterial suspension, adding acetosyringone into the obtained agrobacterium rhizogenes bacterial suspension, and standing; the volume ratio of the primary bacterial suspension to the fresh LB liquid culture medium is 1: 100.

Preferably, in the step S4, the caragana intermedia seedlings which grow for 4-5 days and are good in growth vigor are selected.

Preferably, the culture conditions in step S4 are: the humidity was 90% and the temperature was 28 ℃.

The one-step transformation method further comprises the following steps:

s5, observing the growth condition of the hairy roots of the caragana intermedia, and detecting the expression condition of the e-GFP reporter gene at 14-28 days.

The LB liquid culture medium is prepared according to the following method: taking 1g of Tryptone (Tryptone), 0.5g of Yeast extract (Yeast extract) and 1g of sodium chloride (NaCl), adjusting the pH value to 7.0 by NaOH or HCl, adding deionized water to a constant volume of 100mL, and carrying out autoclaving at 121 ℃ for 20 min.

The LB liquid medium contained 50. mu.g/mL kanamycin and 50. mu.g/mL streptomycin.

The 1/4B5 medium refers to B5 standard medium 1/4 concentration medium.

The OD_600nmThe value is an optical density value measured with the wavelength set at 600nm, is a standard index for tracking the density of microorganisms in a liquid culture, and is generally used to indicate the cell density of bacterial cells.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention transforms the hairy root of the caragana intermedia through a one-step method to ensure that the gene is over expressed in the hairy root, compared with the tissue culture method for transforming the hairy root of the caragana intermedia, the method has simpler operation, does not need to prepare aseptic seedlings, has shorter detection time, high gene expression level and convenient detection, can be directly used for subsequent experiments after the produced hairy root of the plant is detected again, and provides a new way for developing gene function verification and screening functional genes.

2. Compared with the method of stabbing the hypocotyl of the plant by an injection method for hairy root transformation, the one-step transformation method has higher transformation efficiency, the transformation efficiency of the middle caragana hairy root transformed by the method can reach more than 40%, and more hairy roots are generated.

3. In the process of transforming the hairy roots of the caragana intermedia by the one-step method, the invention optimizes the concentration of the bacterial liquid of the agrobacterium rhizogenes and the concentration of the added acetosyringone, and finally shows that the concentration of the bacterial liquid is OD_600nmThe conversion efficiency is optimal when the conversion rate is 0.8-0.95, and the conversion efficiency of the hairy root can be further improved when acetosyringone with the working concentration of 25-100 mu mol/mL is added, so that a foundation is laid for the deep research of the hairy root of the caragana intermedia.

4. In the method, except for infecting the hypocotyl of the caragana intermedia by using the agrobacterium rhizogenes bacterial suspension, the obliquely cut plant hypocotyl is scraped to obtain the agrobacterium rhizogenes bacterial paste, and the step of scraping the bacterial paste is added, so that the conversion power of hairy roots of the caragana intermedia is greatly improved.

5. According to the invention, researches show that the culture medium 1/4B5 is poured into the caragana intermedia seedlings which are planted back to the soil after infection, the hairy roots of the caragana intermedia are more beneficial to grow, meanwhile, the caragana intermedia seedlings are also beneficial to grow, and the caragana intermedia seedlings do not need to be watered within 2 weeks after the culture medium 1/4B5 is poured.

Drawings

FIG. 1 is a schematic structural diagram of a plant expression vector pEGAD;

FIG. 2 shows the infection and cultivation process of caragana intermedia seedlings in the embodiment of the invention: wherein, a figure is a middle caragana seedling growing for 5 days, a figure B is a bevel cutting treatment to the cotyledonary node of the middle caragana seedling, a figure c is a scraped bacterial paste, a figure d is a drawing d in which 1/4B5 culture medium is poured to the middle caragana seedling, a figure e is a drawing in which the middle caragana seedling is cultured in a light incubator, and a figure f is a table diagram of a hairy root of the middle caragana seedling after being transformed for 14 days;

FIG. 3 is a graph of hairy roots of a middle caragana seedling after the middle caragana seedling is infected with Agrobacterium rhizogenes for 14 days (B) and a graph of a middle caragana seedling without Agrobacterium rhizogenes (A) in an example of the present invention;

FIG. 4 is a diagram showing the detection of eGFP in the hairy root of the middle caragana after transformation in the example of the present invention.

Detailed Description

The present invention is described more fully below in order to enable those skilled in the art to better understand the solution of the present invention.

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. In the present specification, "part" and "%" represent "part by mass" and "% by mass", respectively, unless otherwise specified.

The expression vector pEGAD of the invention is from the present of Huazhong agriculture university, and the Agrobacterium rhizogenes K599 is purchased from Shanghai Weidi Biotechnology GmbH.

Example 1

s1, electrically transforming plasmids containing e-GFP expression vectors into agrobacterium rhizogenes K599 competent cells

S11, taking out the pEGAD plasmid, thawing, and placing on ice;

s12, taking out agrobacterium tumefaciens K599 competent cells from a refrigerator at the temperature of-80 ℃, thawing, and placing on ice;

s13, cleaning an electrode cup: soaking the electrode cup in 75% ethanol for 10min, soaking in 100% ethanol for 10min, cleaning with clear water, blow-drying in a fume hood, and pre-cooling;

s14, adding 1 mu L of plasmid pEGAD into competent cells of agrobacterium rhizogenes K599, flicking and uniformly mixing, sucking out all agrobacterium rhizogenes K599 by using a cut and sterilized gun head, and adding the sucked and sterilized agrobacterium rhizogenes K599 into a cleaned and precooled electrode cup (U is 1400V, and T is 5.4-5.8 ms) to obtain a transformation solution;

s15, sucking out all the transformation liquid in the step S14, adding the transformation liquid into a 1.5mL EP tube containing 800 mu L of LB liquid culture medium, and carrying out shaking culture at the oscillation speed of 160rpm at the temperature of 28 ℃ for 2h to obtain a bacterial liquid;

s16, sucking 50 mu L of the bacterial liquid obtained in the step S15, coating the bacterial liquid on a double-resistance plate containing 50 mu g/mL kanamycin and 50 mu g/mL streptomycin, and performing inverted culture at 28 ℃ for 36-48 h;

s17, picking a single colony on the double-resistance plate in the step S16, and identifying whether the expression vector pEGAD is successfully transferred into the agrobacterium rhizogenes K599 by colony PCR.

S2, preparing agrobacterium rhizogenes suspension

S21, inoculating the Agrobacterium rhizogenes K599 identified as containing the plasmid vector pEGAD-e-GFP into 4mL of LB liquid medium (containing 50. mu.g/mL kanamycin and 50. mu.g/mL streptomycin), shaking the bacteria at 28 ℃ and 180rpm overnight, and shaking the bacteria overnight until OD is achieved_600nmThe value is about 0.9, and primary bacterial suspension is obtained;

s22, the next day, preserving a part of the primary bacterial suspension, wherein the preservation steps are as follows: loading 1mL of the primary bacterial suspension into 1.5mL of EP tube, centrifuging at 4000rpm for 2min, discarding the supernatant, adding 800 μ L of fresh LB liquid culture medium and 200 μ L of 75% glycerol, mixing, quick freezing with liquid nitrogen, storing in a refrigerator at-80 deg.C for a long time, and storing the other part of the primary bacterial suspensionThe suspension was expanded, and the portion of the primary suspension was again transferred to a fresh LB liquid medium (containing 50. mu.g/mL kanamycin and 50. mu.g/mL streptomycin), and the suspension was shaken at 28 ℃ and a shaking speed of 180rpm until OD was reached_600nmIs 0.9, and agrobacterium rhizogenes bacterial suspension is obtained; the volume ratio of the primary bacterial suspension to the fresh LB liquid culture medium is 1:100, the primary bacterial suspension is preferably 1mL, and the fresh LB liquid culture medium is preferably 100 mL.

S23, adding acetosyringone with the final concentration of 50 mu mol/mL into the agrobacterium rhizogenes obtained in the step S22 to serve as an inducer, fully and uniformly mixing, and standing for 12 hours in the dark at room temperature, wherein the preparation method of the 50 mu mol/mL acetosyringone is as follows: weighing 0.3924g acetosyringone, dissolving in 10mL DMSO, diluting to 20mL with ultrapure water after completely dissolving, subpackaging into 1mL centrifuge tube, and storing at-20 deg.C for use.

S3, preparing bacterial paste for infection

Centrifuging 1ml of agrobacterium rhizogenes suspension added with acetosyringone, removing part of supernatant, uniformly mixing precipitates, sucking 50 mul of uniformly mixed precipitates, adding the uniformly mixed precipitates into an LB solid culture medium for coating, putting the uniformly mixed precipitates into an incubator at 37 ℃ for culturing for 10-16 hours to prepare bacterial paste for infection, and performing experiments under the room temperature condition of an ultra-clean workbench.

S4. one-step conversion program

S41, planting and selecting middle caragana: weighing special nutrient soil and vermiculite for a laboratory, sterilizing in a sterilizing pot, selecting full and insect-free wild caragana intermedia seeds, sowing the seeds in a pot filled with the nutrient soil and the vermiculite in a ratio of 1:3, putting the pot into a seedling culture plate, covering the pot with a cover, putting the pot into a temperature-controllable illumination incubator, culturing at 28 ℃, growing for 4-5 days, and selecting seedlings with good growth vigor for experiment.

S42, subtracting primary roots of seedlings by using sterilized scissors, beveling the positions about 1cm below cotyledon nodes of the seedlings by using a sterilized blade, increasing the contact area of explants by beveling, discarding the lower parts, soaking the cut in the agrobacterium rhizogenes suspension added with acetosyringone in the step S2 for 1min under a dark environment, scraping the cut to obtain the bacterial paste prepared in the step S3 for infection, wherein the scraped thickness is 0.1-0.5 mm, transplanting the seedlings back into soil, pouring 5ml of 1/4B5 culture medium into each seedling, placing the seedling in a light incubator for culture under the environment of 90% humidity and 28 ℃, and opening a ventilation opening of a seedling tray where caragana middle chick is located after 12-20 days to enable grown hairy roots to gradually adapt to the environment humidity.

S5, observing the growth condition of the hairy roots of the caragana intermedia, pulling out the plants of the hairy roots for detection at 14-28 days, and directly irradiating the positive hairy roots by using a LUYOR-3415 fluorescent protein excitation light source to detect the expression condition of the e-GFP reporter gene.

Example 2

The difference from example 1 is that: the method for preparing the agrobacterium rhizogenes bacterial suspension in the step S2 is as follows: s21, inoculating the agrobacterium rhizogenes K599 identified as containing the plasmid vector pEGAD-e-GFP into 4mL of LB liquid medium (containing 50 mu g/mL of kanamycin and 50 mu g/mL of streptomycin), shaking the bacteria at 28 ℃ at a shaking speed of 180rpm overnight, and shaking the bacteria overnight until the OD600nm value is about 0.9 to obtain a primary bacterial suspension;

s22, the next day, preserving a part of the primary bacterial suspension, wherein the preservation steps are as follows: taking 1mL of the primary bacterial suspension, filling the primary bacterial suspension into a 1.5mL EP tube, centrifuging at the rotating speed of 4000rpm for 2min, discarding the supernatant, adding 800 μ L of fresh LB liquid culture medium and 200 μ L of 75% glycerol, uniformly mixing, quickly freezing by using liquid nitrogen, storing in a refrigerator at minus 80 ℃ for a long time, carrying out amplification culture on the other part of the primary bacterial suspension, transferring 1mL of the primary bacterial suspension into 100mL of fresh LB liquid culture medium again (containing 50 μ g/mL kanamycin and 50 μ g/mL streptomycin), shaking at the shaking speed of 180rpm at 28 ℃ until OD600nm is 0.8, and obtaining the agrobacterium rhizogenes bacterial suspension.

S23, adding acetosyringone with the final concentration of 25 mu mol/mL into the agrobacterium rhizogenes bacterial suspension obtained in the step S22 to serve as an inducer, fully mixing the mixture, and standing the mixture for 12 hours in the dark at room temperature.

Example 3

The difference from example 1 is that: the method for preparing the agrobacterium rhizogenes bacterial suspension in the step S2 is as follows:

s21, identifying the plasmid-containing vectorAgrobacterium rhizogenes K599 of the pEGAD-e-GFP variant was inoculated into 4mL of LB liquid medium (containing 50. mu.g/mL kanamycin and 50. mu.g/mL streptomycin), shaken overnight at 180rpm at 28 ℃ and then cultured overnight until OD_600nmThe value is about 0.9, and primary bacterial suspension is obtained;

s22, the next day, preserving a part of the primary bacterial suspension, wherein the preservation steps are as follows: loading 1mL of the primary bacterial suspension into a 1.5mL EP tube, centrifuging at 4000rpm for 2min, discarding the supernatant, adding 800. mu.L of fresh LB liquid medium and 200. mu.L of 75% glycerol, mixing, quickly freezing with liquid nitrogen, storing in a refrigerator at-80 deg.C for a long time, subjecting the other part of the primary bacterial suspension to amplification culture, transferring 1mL of the primary bacterial suspension into 100mL of fresh LB liquid medium (containing 50. mu.g/mL kanamycin and 50. mu.g/mL streptomycin), shaking at 28 deg.C at 180rpm until OD is OD_600nmTo 0.9, a suspension of Agrobacterium rhizogenes was obtained.

S23, adding acetosyringone with the final concentration of 100 mu mol/mL into the agrobacterium rhizogenes bacterial suspension obtained in the step S22 to serve as an inducer, fully mixing the mixture, and standing the mixture for 12 hours in the dark at room temperature.

Example 4

The difference from example 1 is that: the method for preparing the agrobacterium rhizogenes bacterial suspension in the step S2 is as follows: s21, inoculating the Agrobacterium rhizogenes K599 identified as containing the plasmid vector pEGAD-e-GFP into 4mL of LB liquid medium (containing 50. mu.g/mL kanamycin and 50. mu.g/mL streptomycin), shaking the bacteria at 28 ℃ and 180rpm overnight, and shaking the bacteria overnight until OD is achieved_600nmThe value is about 0.9, and primary bacterial suspension is obtained;

s22, the next day, preserving a part of the primary bacterial suspension, wherein the preservation steps are as follows: loading 1mL of the primary bacterial suspension into a 1.5mL EP tube, centrifuging at 4000rpm for 2min, discarding the supernatant, adding 800. mu.L of fresh LB liquid medium and 200. mu.L of 75% glycerol, mixing, quickly freezing with liquid nitrogen, storing in a refrigerator at-80 deg.C for a long time, subjecting the other part of the primary bacterial suspension to amplification culture, transferring 1mL of the primary bacterial suspension into 100mL of fresh LB liquid medium (containing 50. mu.g/mL kanamycin and 50. mu.g/mL streptomycin), shaking at 28 deg.C at 180rpm until OD is OD_600nmIs 0.9And 5, obtaining the agrobacterium rhizogenes suspension.

S23, adding acetosyringone with the final concentration of 50 mu mol/mL into the agrobacterium rhizogenes bacterial suspension obtained in the step S22 to serve as an inducer, fully mixing the mixture evenly, and standing the mixture for 12 hours in the dark at room temperature.

Comparative example 1

The difference from example 1 is that: the one-step conversion procedure in step S4 is as follows:

S42, subtracting the primary root of the seedling by using sterilized scissors, beveling the position about 1cm below the cotyledon node of the seedling by using a sterilized blade, discarding the lower part, soaking the cut in the agrobacterium rhizogenes bacterial suspension prepared in the step S2 for 1min, transplanting the seedling back to the soil, pouring 5ml of 1/4B5 culture medium into each seedling, and placing the seedling in an illumination culture box for culture under the environment with the humidity of 90% and the temperature of 28 ℃.

Comparative example 2

S42, injecting the agrobacterium rhizogenes suspension prepared in the step S2 to cotyledonary nodes of seedlings, transplanting the seedlings back to soil after infection, pouring 5ml of 1/4B5 culture medium to each seedling, and placing the seedlings in a light incubator to culture in an environment with the humidity of 90% and the temperature of 28 ℃.

According to researches, 1/4B5 culture medium is poured into the caragana intermedia seedlings which are planted back to the soil after infection, the hairy roots of the caragana intermedia are more favorably grown, meanwhile, the caragana intermedia seedlings are also favorably grown, and the caragana intermedia seedlings do not need to be watered within 2 weeks after the 1/4B5 culture medium is poured.

After examining the caragana sylvestris plants with long hairy roots 14 days after the impregnation in examples 1 to 4 and comparative examples to 2, it was found that the OD was when_600nmWhen the concentration is 0.9 and the final concentration of the added acetosyringone is 50 mu mol/L, the induction rate of the hairy roots is the highest and can reach more than 40 percent. In contrast, in the transformation method in which the cotyledon nodes of the seedlings in the comparative example 1 are only soaked in the agrobacterium rhizogenes suspension and are not scraped and infected with the bacterial paste, the transformation rate of the hairy roots of the caragana intermedia is lower than that in the transformation method in the example 1. The conversion rate of the injection method for transforming the hairy root of the caragana intermedia in the comparative example 2 is much lower than that of the hairy root of the caragana intermedia in the example 1, and the conversion rate of the injection method for transforming the hairy root of the caragana intermedia only reaches about 30 percent.

As can be seen from FIGS. 3 and 4, the intermediate caragana plant obtained by the one-step transformation method of the intermediate caragana hairy root of the present invention can grow hairy root after being dip-dyed for 14 days, and the positive hairy root is directly irradiated by the excitation light source of LUYOR-3415 fluorescent protein, so that the positive hairy root can see strong fluorescence. The fluorescence signal of the transformed e-GFP hairy root is obviously stronger than that of the normal root observed under a fluorescence microscope.

The method for transforming the hairy root of the caragana intermedia by using the one-step transformation method has the advantages that the inductivity can reach more than 40 percent, and compared with the method for transforming the hairy root of the caragana intermedia by using an injection method, the method has shorter time and higher efficiency.

The above-described embodiments are preferred implementations of the present invention, and the present invention may be implemented in other ways without departing from the spirit of the present invention.

Claims

1. An agrobacterium rhizogenes-mediated one-step transformation method for hairy roots of middle caragana, which is characterized by comprising the following steps:

2. The one-step transformation method of claim 1, wherein the e-GFP reporter gene-containing expression vector is pEGAD.

3. The one-step transformation method according to claim 1, wherein the OD of the Agrobacterium rhizogenes suspension in step S2_600nmThe value was 0.9 and the final acetosyringone concentration was 50. mu. mol/mL.

4. The one-step transformation method according to claim 1, wherein the bacterial paste used in the step S3 is infected under culture conditions: the temperature of the incubator is set to 37 ℃, and the culture time is 10-16 hours.

5. The one-step transformation method of claim 1, wherein the soaking time of the middle caragana seedling cotyledonary node cut in step S4 is 1min, and the thickness of the bacterial paste at the cut is 0.1-0.5 mm.

6. The one-step transformation method of claim 1, wherein the 1/4B5 medium is used in an amount of 5mL in step S4.

7. The one-step transformation method of claim 1, wherein the specific steps of step S2 are as follows: inoculating the Agrobacterium rhizogenes K599 obtained in the step S1 to LB liquid medium, and culturing at 28 ℃ with shaking to OD_600nmObtaining a primary bacterial suspension for 0.9, then transferring the primary bacterial suspension into a fresh LB liquid culture medium again, and carrying out shake culture on the primary bacterial suspension to OD at 28 DEG C_600nmAfter the concentration is 0.8-0.95, obtaining agrobacterium rhizogenes bacterial suspension, adding acetosyringone into the obtained agrobacterium rhizogenes bacterial suspension, and standing; the volume ratio of the primary bacterial suspension to the fresh LB liquid culture medium is 1: 100.

8. The one-step transformation method of claim 1, wherein in step S4, the caragana intermedia seedlings which grow for 4-5 days and have good growth vigor are selected.

9. The one-step transformation method according to claim 1, wherein the culture conditions in step S4 are: the humidity was 90% and the temperature was 28 ℃.

10. The one-step conversion process of claim 1, further comprising the steps of: