A method for genetic transformation of robinia hispida hairy root mediated by agrobacterium rhizogenes

Abstract

The application discloses a genetic transformation method of Robinia pseudoacacia hairy roots mediated by Agrobacterium rhizogenes, and belongs to the technical field of plant genetic engineering and molecular breeding. The method comprises the following steps: preparing an Agrobacterium rhizogenes infection solution carrying a target gene; taking a Robinia pseudoacacia seedling, removing the root at the root stem connection to obtain an explant; immersing the wound of the explant into the infection solution to perform infection; subsequently, directly planting the infected explant into a substrate to perform co-culture and root induction until the transgenic hairy roots are formed at the wound. The method provided by the application completely abandons aseptic tissue culture and a complicated plant regeneration process, and realizes genetic transformation of Robinia pseudoacacia under non-aseptic and non-detached conditions. The method can shorten the transformation period from several months to 3-4 weeks, and the positive transformation rate can be stabilized to be more than 60%. The method is simple in operation, low in cost, efficient and stable, and can provide important technical support for functional genomics research, genetic improvement and molecular breeding of Robinia pseudoacacia.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology, specifically to the field of plant genetic engineering and molecular breeding technology. In particular, this invention relates to a method utilizing *Agrobacterium rhizogenes* (…). Agrobacterium rhizogenes ) as a mediator, under non-in vitro, non-sterile environmental conditions, for the important woody plant Robinia pseudoacacia ( Robinia pseudoacacia A method for rapid and efficient genetic transformation of L. (L.) that can be used to induce the production of transgenic hairy roots. Background Technology

[0002] locust( Robinia pseudoacacia Black locust (L.) belongs to the genus *Robinia* in the family Leguminosae and is a tall, deciduous tree. It is characterized by rapid growth, strong adaptability, drought and poor soil tolerance, and excellent wood quality. Its roots can form a symbiotic relationship with rhizobia and perform biological nitrogen fixation, effectively improving soil conditions. Therefore, black locust is widely used in the construction of fast-growing and high-yield forests, soil and water conservation, urban and rural greening, and energy forest development, and is an important economic and ecological tree species widely introduced and cultivated globally.

[0003] Currently, genetic transformation of Robinia pseudoacacia mainly relies on traditional tissue culture regeneration systems. These systems typically use leaves or stem segments as explants, obtaining transgenic plants through callus induction, adventitious bud differentiation, and plant regeneration. However, this approach suffers from low transformation efficiency, long cycles, complex procedures, and strong genotype dependence, severely hindering the development of functional gene research and molecular breeding in Robinia pseudoacacia. Furthermore, traditional genetic transformation requires multiple culture stages, including explant dedifferentiation and redifferentiation, with the entire transformation cycle usually lasting over six months. It also demands high levels of aseptic culture conditions, resulting in high technical barriers, significant labor input, and high culture costs.

[0004] Agrobacterium rhizogenes ( Agrobacterium rhizogenes Agrobacterium rhizogenes-mediated hairy root induction is a highly efficient plant genetic transformation method. After infecting plants, Agrobacterium rhizogenes integrates its root-inducing plasmid's T-DNA into the plant genome, inducing the production of numerous highly branched hairy roots at the infected site. These hairy roots are characterized by rapid growth, genetic stability, and strong hormone autonomy, and can grow rapidly on hormone-free media. This technology offers advantages such as ease of operation, short cycle time, and high transformation efficiency, and has been widely used in various plants including legumes, solanaceae, and cruciferous families. Hairy root culture systems provide effective tools for functional gene verification, regulation of secondary metabolite synthesis, and research on root development mechanisms.

[0005] However, as a woody leguminous plant, the black locust exhibits significant differences in tissue structure and physiological characteristics compared to herbaceous plants. Issues such as explant characteristics, Agrobacterium tumefaciens compatibility, co-culture conditions, hairy root induction efficiency, and subsequent stability are intertwined. Systematic research and reports on Agrobacterium tumefaciens-mediated hairy root induction technology under non-sterile conditions for black locust have yet to be found. Therefore, establishing an efficient, stable, and easy-to-operate genetic transformation system for black locust hairy roots is of great significance for advancing gene function research and genetic improvement in black locust. Summary of the Invention

[0006] The purpose of this invention is to provide a method for genetic transformation of hairy roots of Robinia pseudoacacia mediated by Agrobacterium rhizogenes and its application, overcoming the problems of low efficiency, long cycle and complicated operation of genetic transformation of Robinia pseudoacacia in the prior art, and establishing a genetic transformation system mediated by Agrobacterium rhizogenes suitable for Robinia pseudoacacia, thereby improving the hairy root induction rate and genetic transformation rate of Robinia pseudoacacia.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for genetic transformation of hairy roots of Robinia pseudoacacia mediated by Agrobacterium rhizogenes under non-sterile conditions, comprising the following steps: introducing an expression vector into Agrobacterium rhizogenes and mixing it with an infection solution to prepare an Agrobacterium rhizogenes infection solution; cutting off the roots of Robinia pseudoacacia seedlings within 2 mm from the root-stem junction to the lower end of the hypocotyl, and then immersing them in the Agrobacterium rhizogenes infection solution for 0.5-2 h; after infection, inserting the Robinia pseudoacacia seedlings into a non-sterile growth substrate and dark-culturing them for 36 h at a relative humidity of 80%, then transferring them to a light intensity of 10000 lx, a light / dark cycle of 16 h / 8 h, and a relative humidity of 70% for continued culture until hairy roots are produced; the OD of the Agrobacterium rhizogenes infection solution is... 600 The value is 0.5 to 2.0.

[0008] Preferably, the infection solution is formulated as follows: 10 mM MES, 10 mM MgCl2, 15 mM D-glucose and 200 μM AS, and the pH of the buffer solution is adjusted to 5.6 with 1 mol / L KOH.

[0009] Preferably, the Agrobacterium rhizogenes is Agrobacterium rhizogenes K599.

[0010] Preferably, healthy, uniformly growing black locust seedlings are selected, and seedlings that have grown for 6 days are preferred.

[0011] Preferably, the expression vector comprises the pBI121-eGFP vector carrying the eGFP marker gene.

[0012] Preferably, the culture medium for Agrobacterium rhizogenes is TY medium.

[0013] Preferably, the non-sterile growth substrate is peat moss: perlite = 5:1.

[0014] The present invention also provides the application of the method in improving the rooting rate and / or genetic transformation rate of hairy roots of Robinia pseudoacacia.

[0015] The present invention also provides for the application of the method in any of the following: S1. Rapid genetic transformation of black locust hairy roots mediated by Agrobacterium rhizogenes under non-sterile environmental conditions; S2. Shorten the genetic transformation cycle; S3, Research on functional genes of Robinia pseudoacacia; S4. Robinia pseudoacacia genetic breeding.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention is the first to develop a method for rapid genetic transformation of hairy roots of Robinia pseudoacacia mediated by Agrobacterium rhizogenes under non-sterile conditions. This invention systematically screens the infection solution concentration (OD). 600 The effects of OD values ​​(0.5, 1.0, 1.5, 2.0) and infection time (0.5 h, 1 h, 1.5 h, 2 h) on the induction of hairy roots in Robinia pseudoacacia were investigated, and the optimal transformation conditions were finally determined to be: using OD... 600 With an infection solution of 1.5, after 1 hour of infection, the efficiency of inducing transgenic hairy roots of black locust can reach up to 63.16%.

[0017] Compared with traditional Robinia pseudoacacia genetic transformation systems that rely on tissue culture conditions, this system has the following significant advantages: (1) It is simple to operate and does not require a complex tissue culture process; (2) It has a short cycle, requiring only 3-4 weeks from infection to obtaining transgenic hairy roots, which is 3-4 months shorter than traditional methods; (3) It is low-cost and can be carried out under non-sterile conditions without expensive equipment and reagents; (4) It is highly efficient, with a transformation rate of over 60%; (5) It allows for visual screening, using eGFP marker genes to observe phenotypes under a portable blue excitation light source to determine whether the transformation was successful. This invention realizes a simple, efficient, and low-cost genetic transformation of Robinia pseudoacacia, which not only provides a new approach for the genetic transformation of Robinia pseudoacacia, but also lays a solid foundation for the study of gene function, the analysis of the regulatory mechanism of secondary metabolites, and molecular breeding of Robinia pseudoacacia. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the overall technical flow of the method described in this invention.

[0019] Figure 2 This is a schematic diagram of the plasmid map of the pBI121-eGFP binary expression vector used in the embodiments of the present invention. The key elements such as the eGFP expression frame and selection marker gene in the T-DNA region are clearly marked in the figure.

[0020] Figure 3 These are comparative photographs showing the effects of implementing the method of the present invention. The left image shows normal roots growing from untransformed black locust seedlings in the substrate, observed under a portable blue light excitation source; the right image shows transgenic hairy roots, successfully induced at the cut site and expressing eGFP with bright green fluorescence, observed under a portable blue light excitation source after treatment by the method of the present invention.

[0021] Figure 4 Comparison of DNA extracted from untransformed black locust roots and transgenic positive roots obtained in Example 1, followed by PCR detection of the target gene. Detailed Implementation

[0022] This invention is the first to develop a method for rapid genetic transformation of hairy roots of Robinia pseudoacacia mediated by Agrobacterium rhizogenes under non-sterile conditions, comprising the following steps: introducing an expression vector into Agrobacterium rhizogenes and mixing it with an infection solution to prepare an Agrobacterium rhizogenes infection solution; cutting off the roots of Robinia pseudoacacia seedlings within 2 mm from the root-stem junction to the lower end of the hypocotyl, and then immersing them in the Agrobacterium rhizogenes infection solution for 0.5-2 h; after infection, the Robinia pseudoacacia seedlings are planted in a substrate for dark culture and continue to grow until hairy roots are produced; the OD of the Agrobacterium rhizogenes infection solution containing the target gene is measured. 600 The value is 0.5 to 2.0.

[0023] In this invention, the expression vector contains the target gene to be expressed. The target gene refers to a gene related to Robinia pseudoacacia functional gene research or Robinia pseudoacacia genetic breeding, and is not specifically limited to this gene. As an optional implementation, the expression vector is a pBI121-eGFP vector carrying an eGFP marker gene. The eGFP marker gene carried by this vector is a visual reporter gene; its phenotype can be observed under a portable blue light excitation source, allowing for direct judgment of whether the gene has been successfully transformed and expressed. In this invention, the *Agrobacterium rhizogenes* is preferably *Agrobacterium rhizogenes* K599. The pBI121-eGFP vector is transformed into competent *Agrobacterium rhizogenes* cells and cultured in a culture medium to prepare *Agrobacterium rhizogenes* bacterial suspension. The culture medium is preferably TY liquid medium. The OD of the *Agrobacterium rhizogenes* bacterial suspension... 600 =0.8~1.0.

[0024] In this invention, the method for preparing the *Agrobacterium rhizogenes* infection solution includes suspending the bacterial cells after centrifuging the prepared *Agrobacterium rhizogenes* K599 bacterial suspension in the infection solution. The infection solution has the following formulation: 10 mM MES (morpholine ethanesulfonic acid), 10 mM MgCl2 (magnesium chloride), 15 mM D-glucose, and 200 μM AS (acetylsyl eugenol). The pH of the buffer solution is adjusted to 5.6 with 1 mol / L KOH. When suspending the bacterial cells in the infection solution, the OD value of the *Agrobacterium rhizogenes* infection solution is measured. 600 The value is 0.5 to 2.0; more preferably 1.5.

[0025] In this invention, the infection time is preferably 0.5 to 2 hours, more preferably 1 hour; after infection, the black locust seedlings are planted in a substrate for dark culture and continue to grow until hairy roots are produced; the temperature of the dark culture is 20 to 24 ℃, more preferably 22 ℃, and the dark culture time is preferably 36 hours.

[0026] As an optional implementation method, the specific steps of the Agrobacterium rhizogenes-mediated genetic transformation method for hairy roots of Robinia pseudoacacia are as follows: S1. Explant preparation: Select healthy, uniformly growing black locust seedlings (preferably seedlings grown for 6 days). Using a sharp, sterile blade, cut the black locust seedling at the root-stem junction, within 2 mm from the root-stem junction to the lower end of the hypocotyl, remove its original root system, and use the cut hypocotyl portion as the explant to be transformed; S2. Chemical transformation of Agrobacterium rhizogenes: The pBI121-eGFP vector was transformed into Agrobacterium rhizogenes K599 competent cells using the liquid nitrogen freeze-thaw method. The cells were then plated on TY solid medium containing 50 mg / L kanamycin and cultured. Single colonies were picked for colony PCR identification to obtain positive strains. S3. Preparation of bacterial culture: The bacterial culture of the positive strain *Agrobacterium rhizogenes* was streaked onto solid TY medium containing the corresponding antibiotic and incubated at 28 ℃ for 2 days to obtain single colonies. Single colonies were picked and placed in TY liquid medium containing 50 mg / L kanamycin and activated at 28 ℃ and 200 rpm for 12-20 h using a shaker. The bacterial concentration (OD) was then measured. 600 When the value reaches 0.8~1.0, the culture is stopped, and Agrobacterium rhizogenes bacterial solution is obtained; S4. Preparation of Infection Solution: After centrifuging the *Agrobacterium rhizogenes* bacterial suspension obtained in step S3 at 5000 rpm for 5 min, collect the bacterial precipitate and resuspend it in an infection solution containing 10 mM MES, 10 mM MgCl2, 15 mM D-glucose, and 200 μM AS at pH 5.6. Adjust the concentration of the infection solution to OD. 600 It is 1.5; S5. Agrobacterium rhizogenes infection: The infection recipient material from step S1 was placed in the infection solution of S4 and allowed to stand for 1 h under normal temperature, normal pressure, and dark conditions. After infection, the seedlings were planted in the substrate and cultured in the dark for 36 h at 22 ℃ and 80% relative humidity. S6. Transgenic hairy root induction and identification: The material after dark culture was transferred to a light intensity of 10000 lx and a photoperiod of 16 h / 8 h. During the period, the soil substrate moisture content was kept greater than 70%. Transgenic hairy roots of Robinia pseudoacacia were obtained by continuing culture. The present invention also provides the application of the method in improving the rooting rate and genetic transformation rate of hairy roots of black locust.

[0027] The present invention also provides the application of the method in any of the following: S1. Rapid genetic transformation of black locust hairy roots mediated by Agrobacterium rhizogenes under non-sterile environmental conditions; S2. Shorten the genetic transformation cycle; S3, Research on functional genes of Robinia pseudoacacia; S4. Robinia pseudoacacia genetic breeding.

[0028] In the following embodiments of the present invention, the pBI121-eGFP vector was long-term preserved in our laboratory, and the Agrobacterium rhizogenes K599 competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0029] In the following embodiments of the present invention, the TY culture medium is prepared by adding 5 g of peptone and 3 g of yeast extract to an Erlenmeyer flask, adding water to a volume of 1 L, completely dissolving, and then autoclaving at 121 °C for 20 min; a 1 M calcium chloride aqueous solution is prepared and autoclaved at 121 °C for 20 min, and 10 mL of sterile 1 M calcium chloride aqueous solution is added to every 1 L of sterilized TY liquid culture medium. If preparing TY solid culture medium, 5 g of tryptone, 3 g of yeast extract, and 15 g of agar are added to 1 L of culture medium, and after sterilization, 10 mL of sterile 1 M calcium chloride aqueous solution is added. In the following embodiments of the present invention, the basic infection solution is formulated as follows: 1 mL of 1 M MgCl2, 2 mL of 0.5 M MES, 1.5 mL of 1 M D-glucose, 0.2 mL of 0.1 M AS, 96.8 mL of ddH2O, pH 5.6, totaling 100 mL.

[0030] In the following embodiments of the present invention, the soil matrix is ​​peat moss: perlite = 5:1.

[0031] Unless otherwise specified, the test methods used in the following examples are conventional test methods; the materials and reagents used are commercially available unless otherwise specified.

[0032] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments thereof.

[0033] Example 1: Agrobacterium rhizogenes-mediated genetic transformation of Robinia pseudoacacia 1. Explant preparation Select healthy, uniformly growing black locust seedlings that have been growing for 6 days. Use a sharp, sterile blade to remove the original roots at the root-stem junction, within a range of 2 mm from the root-stem junction to the lower end of the hypocotyl. Use the hypocotyl portion with the cut as the explant to be transformed.

[0034] 2. Preparation of infection solution for Agrobacterium rhizogenes K599 This embodiment provides preliminary experimental conditions, using OD. 600 =1.0, infection duration 1.5 h. Agrobacterium rhizogenes K599 bacterial suspension carrying pBI121-eGFP plasmid was streaked onto TY solid medium containing 50 mg / L kanamycin to obtain single colonies of Agrobacterium K599. Single colonies of Agrobacterium K599 were picked and activated in TY liquid medium containing 50 mg / L kanamycin at 28 ℃ and 200 rpm for 12 h to obtain activated Agrobacterium K599 bacterial suspension. 500 μL of the activated Agrobacterium K599 bacterial suspension was added to 100 mL of TY liquid medium containing 50 mg / L kanamycin and activated by shaking at 28 ℃ and 200 rpm for 12-20 h until OD was reached. 600 The culture was stopped when the concentration of *Agrobacterium rhizogenes* reached 0.8–1.0, yielding the bacterial suspension. The bacterial suspension was centrifuged at 5000 rpm for 5 min to collect the cells, which were then resuspended in the infection solution to OD values. 600 =1.0, let stand in the dark for 2.5 h to obtain Agrobacterium rhizogenes infection solution.

[0035] 3. Infection and Co-cultivation The black locust explants were immersed in different concentrations of Agrobacterium rhizogenes infection solution and allowed to stand for 1.5 h under room temperature, normal pressure, and dark conditions. After infection, the seedlings were planted in moist substrate and cultured in the dark for 36 h at 22 ℃ and 80% relative humidity. Then, they were transferred to light (22 ℃, 16 h light; 22 ℃, 8 h dark) for induction, with a light intensity of 10000 lx and a relative humidity of 70%. The hairy root induction was observed every 3 days, and the transgenic root induction was tested after 2 weeks.

[0036] 4. Detection of genetically modified roots The roots obtained in step 3 were tested for green fluorescence using a Luyang fluorescent flashlight, and the results are as follows: Figure 3 The images shown are photographs of the hair roots of the CK control group (CK being explants with the hypocotyl removed but without infection) and the eGFP fluorescent tag group, respectively, as detected by Lu Yang fluorescent flashlight.

[0037] DNA was extracted from the roots of the control (CK) and transgenic roots respectively (using the Polysaccharide-Polyphenol Plant Genomic DNA Extraction Kit (DP360) from Tiangen Biotech (Beijing) Co., Ltd.). The target eGFP gene was cloned by PCR, where eGFP-F: 5'-atggtgagcaagggcgagg-3'; eGFP-R: 5'-gttgtggctgttgtagttgtactcc-3'. The corresponding bands were confirmed to be amplified in the transgenic hairy roots. (See attached data for the detection results.) Figure 4 In the figure, lines 1-3 represent three different transgenic root systems. It can be seen that the CK control group did not have the eGFP target gene band, while the control plasmid and the transgenic root system contained the eGFP target gene band.

[0038] According to the test results in this embodiment, the K599 Agrobacterium rhizogenes infection of the hypocotyl of Robinia pseudoacacia seedlings showed that the seedlings began to grow hairy roots after 6 days, and the rooting efficiency (represented by the number of seedlings with roots / the number of infected explants) was 70% after 2 weeks, and the transgenic efficiency (represented by the number of positive transgenic plants / the number of seedlings with roots) was 28%.

[0039] Example 2: Screening of the optimal induction system for hairy roots of Robinia pseudoacacia This embodiment 2 is performed in accordance with the operation of embodiment 1, except that: Different infection concentrations are: containing OD 600 =0.5, OD 600 =1.0, OD 600 =1.5 and OD 600 =2.0% Agrobacterium rhizogenes K599 infection solution; different infection durations: 0.5 h, 1 h, 1.5 h and 2 h.

[0040] Table 1. Effects of different experimental treatments on hairy root induction and transformation of Robinia pseudoacacia.

[0041] As shown in Table 1, the optimal induction conditions for hairy roots of Robinia pseudoacacia are: using OD 600Infecting explants with Agrobacterium rhizogenes K599 at a concentration of 1.5 for 1 h showed good effects on the induction and genetic transformation of hairy roots in Robinia pseudoacacia, with a hairy root occurrence rate of 63.33% and a positive rate of 63.16%. Therefore, it is the best choice for the genetic transformation of hairy roots in Robinia pseudoacacia.

[0042] In summary, this invention screened the infection time and infection concentration conditions, and finally concluded that OD... 600 A 1.5 μL inoculum solution, inoculated for 1 hour, can induce hairy roots in *Robinia pseudoacacia* with an efficiency exceeding 60%. Compared to tissue culture-dependent genetic transformation systems, this system significantly shortens the transformation cycle, enabling the production of transgenic material in large quantities within a short time, achieving simple, efficient, and low-cost genetic transformation of *Robinia pseudoacacia*. This invention not only provides a new approach for the genetic transformation of *Robinia pseudoacacia* but also lays the foundation for gene function research and molecular breeding in *Robinia pseudoacacia*. Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for genetic transformation of hairy roots of Robinia pseudoacacia mediated by Agrobacterium rhizogenes, characterized in that, Includes the following steps: a) Explant preparation: Select Robinia pseudoacacia ( Robinia pseudoacacia L.) seedlings, their roots are removed by horizontal transverse cuts at the rhizome junction to obtain hypocotyl explants with wounds; b) Agrobacterium rhizogenes carrying the exogenous target gene ( Agrobacterium rhizogenes A system for infection was prepared, and the wound site of the explant was brought into contact with the infection system to allow Agrobacterium rhizogenes to infect the plant tissue; c) The infected explants were directly transferred to a non-sterile solid growth substrate, and hairy roots were induced at the infected site without the need for tissue culture and exogenous hormones. d) During the culture process, hairy roots of Robinia pseudoacacia that stably express the exogenous target gene were obtained from the explant wound.

2. The method according to claim 1, characterized in that, The method described is applicable to the genetic transformation of Robinia pseudoacacia, a woody leguminous plant that is prone to explant browning and has difficulty in callus differentiation.

3. The method according to claim 1, characterized in that, The black locust seedlings mentioned in step a) are seed seedlings that have grown for 6 days under light conditions after seed germination. Their roots are removed, and the cutting position is limited to within 2 mm from the junction of the rootstock to the lower end of the hypocotyl, avoiding excessive cutting upwards and damaging the upper part of the hypocotyl tissue, so as to obtain hypocotyl explants with neat wounds.

4. The method according to claim 1, characterized in that, The Agrobacterium rhizogenes mentioned in step b) is Agrobacterium rhizogenes K599 or an engineered strain with equivalent infectivity.

5. The method according to claim 1, characterized in that, The infection solution in step b) is formulated as follows: 10 mM MMES, 10 mM MgCl2, 15 mM D-glucose and 200 μM AS, with the pH of the buffer adjusted to 5.6 using 1 mol / L KOH.

6. The method according to claim 1, characterized in that, The optical density (OD) of the *Agrobacterium rhizogenes* infection solution described in step b) at a wavelength of 600 nm. 600 The value is 0.5~2.

0.

7. The method according to claim 1, characterized in that, The infection treatment method described in step b) involves completely immersing the wound site of the explant in Agrobacterium rhizogenes infection solution and allowing it to stand and soak for 0.5 to 2 hours under normal temperature, pressure, and dark conditions to complete the infection treatment.

8. The method according to claim 1, characterized in that, The cultivation process described in step c) includes: first, planting the explants into a non-sterile solid growth substrate and then co-culturing them for 36 h in the dark at 22 ℃ and 80% relative humidity. Then, transferring them to a light intensity of 10000 lx, a light / dark cycle of 16 h / 8 h, and 70% relative humidity for further cultivation. The non-sterile solid growth substrate is peat moss: perlite = 5:

1.

9. The method according to claim 1, characterized in that, The target gene expression vector also contains a reporter gene; the reporter gene is preferably a fluorescent protein gene, and a portable fluorescent excitation light source is used to irradiate the hairy roots to detect whether they express fluorescence, so as to identify positive transformants.

10. The method according to claim 1, characterized in that, Following step d), a step of molecular verification of the positive transgenic hairy roots is included, which includes PCR amplification using primers specific to the reporter gene to confirm successful integration of the exogenous gene.

Images