A method for inducing transgenic roots of Lycium barbarum mediated by Agrobacterium rhizogenes

Abstract

This invention belongs to the field of plant genetic engineering technology, specifically relating to a method for inducing transgenic roots of Lycium barbarum mediated by Agrobacterium rhizogenes, comprising the following steps: S1, preparation of sterile explants of Lycium barbarum; S2, preparation of infecting bacterial solution; S3, pretreatment and negative pressure inoculation; S4, co-culture and gradient sterilization; S5, delayed culture and gradient resistance screening; S6, identification of positive hairy roots and root strengthening culture. This invention uses semi-lignified root-cutting stem segments with axillary buds as explants. The wound surface formed by the 45° oblique cut increases the infection site of Agrobacterium. Combined with negative pressure assisted inoculation technology, the transformation efficiency of Agrobacterium is significantly improved. Experimental verification shows that the induction rate of hairy roots of Lycium barbarum in this invention can reach more than 85%, and the positive transformation rate can reach more than 70%, which is far higher than the average level of 30-40% of the prior art.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a method for inducing transgenic roots of wolfberry mediated by Agrobacterium rhizogenes. Background Technology

[0002] Goji berry (Lycium barbarum L.) is a perennial deciduous shrub endemic to my country, used both as food and medicine. Ningxia goji berry is the only original species of goji berry medicinal material included in the Chinese Pharmacopoeia, possessing extremely high economic value and application prospects in the fields of medicine, food, and health products. Currently, most of the main goji berry varieties cultivated in my country are traditionally bred varieties, which generally suffer from weak resistance (insufficient resistance to salt, drought, and root rot), limited quality traits, and bottlenecks in yield improvement, severely restricting the high-quality development of the goji berry industry.

[0003] Genetic engineering breeding is a core technology for breaking down genetic barriers and targeted improving the agronomic traits of wolfberry. An efficient and stable genetic transformation system is fundamental to achieving precise gene editing and the introduction of exogenous genes in wolfberry. Currently, research on wolfberry genetic transformation is still in its early stages. Existing technologies mostly use wolfberry leaves, hypocotyls of sterile seedlings, and ordinary stem segments as explants for Agrobacterium transformation, which has the following core defects: First, the explant regeneration rate is low and the transformation efficiency is poor, with most systems showing a hairy root induction rate of less than 40% and a positive transformation rate of less than 30%. Second, the chimerism rate is high, and the stability of exogenous gene integration is poor, making it difficult to obtain homozygous transgenic materials. Third, there is strong genotype dependence, effective only for a single wolfberry variety and unable to adapt to large-scale transformation of the main cultivated varieties. Fourth, the screening process is unreasonable, with a high false positive rate, and a standardized regeneration system from hairy roots to complete plants has not been established, resulting in a transformation cycle of more than 6 months.

[0004] Agrobacterium rhizogenes can induce hairy roots in plants, offering advantages such as high transformation efficiency, good genetic stability, and simple operation. It is an ideal tool for verifying the function of root-related genes and regulating the synthesis of secondary metabolites. For wolfberry, the root system is a key organ for nutrient absorption, response to abiotic stress, and synthesis of core functional components. Establishing an efficient, stable, and universal Agrobacterium rhizogenes-mediated transgenic root induction system for wolfberry has significant theoretical and practical implications for the control of soil-borne diseases and pests, improvement of root traits, and gene function discovery in wolfberry.

[0005] Currently, the publicly available Agrobacterium rhizogenes transformation technologies for wolfberry are mostly limited to the transformation and verification of specific genes. For example, CN202410255585.5 discloses an Agrobacterium rhizogenes-mediated method for transforming Ningxia wolfberry into the LbbHLH36 gene, but its protection scope is limited to the transformation of specific genes and a universal standardized transformation system has not been established. Moreover, the existing technologies have not been systematically optimized for wolfberry explant types, infection methods, screening systems, and plant regeneration, and cannot solve the core pain points of low efficiency, many chimeras, and strong genotype dependence of the existing transformation system. Summary of the Invention

[0006] The purpose of this invention is to provide a method for inducing transgenic roots of Lycium barbarum mediated by Agrobacterium rhizogenes. This method aims to provide a high-efficiency, genetically stable, chimeric, and genotype-dependent method for inducing transgenic roots of Lycium barbarum mediated by Agrobacterium rhizogenes, with standardized operation procedures. It also provides the application of this method in the genetic improvement of Lycium barbarum, providing core technical support for the verification of Lycium barbarum gene function and the creation of new transgenic varieties.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for inducing transgenic roots of Lycium barbarum mediated by Agrobacterium rhizogenes, comprising the following technical solution.

[0008] S1. Preparation of sterile explants of wolfberry: obtain sterile tissue culture seedlings of wolfberry, cut semi-lignified stem segments with axillary buds, and prepare explants with root severance wounds; S2. Preparation of infecting bacterial solution: Transform the plant binary expression vector carrying the target exogenous gene into Agrobacterium rhizogenes to prepare the activated infecting bacterial solution; S3. Pretreatment and negative pressure infection: The explants were pre-cultured in a pretreatment solution containing acetylsuccinone, and then infected with Agrobacterium using a negative pressure-assisted infection method. S4. Co-culture and gradient sterilization: The infected explants were placed in a co-culture medium and cultured in the dark. After completion, the Agrobacterium tumefaciens was sterilized by gradient immersion. S5. Delayed culture and gradient resistance screening: After sterilization, the explants were first subjected to antibiotic-free delayed culture, and then cultured in stages using resistance screening medium with increasing concentration gradients to induce the formation of positive hairy roots. S6. Identification of positive hairy roots and root-strengthening culture: Positive transgenic hairy roots were screened by fluorescent labeling and molecular biological identification, and then inoculated into root-strengthening culture medium to complete propagation and root strengthening. Furthermore, the present invention optimizes the core parameters and culture medium formulation of each step, as follows: Explant preparation: Select mature wolfberry seeds, surface disinfect with 75% ethanol for 1 min, rinse twice with sterile water, disinfect with 2% sodium hypochlorite solution for 7 min, rinse 5-6 times with sterile water, absorb the water and inoculate into 1 / 2 MS basic medium (30 g / L sucrose + 7.5 g / L agar, pH 5.8), culture for 14 days at 25±1℃, 16 h / d light, and 6000 lx light intensity to obtain sterile tissue culture seedlings with a height of 10-12 cm and 12-15 true leaves; cut semi-lignified stem segments with 1-2 plump axillary buds and a length of 3-4 cm from the base of the sterile seedlings, make a 45° oblique cut at the lower end of the stem segment to form a root severance infection wound, and make a horizontal cut at the upper end, retaining 1 healthy true leaf to obtain the explant.

[0009] Preparation of infecting bacterial suspension: Agrobacterium rhizogenes strain K599 was used, and the plant binary expression vector was the pCAMBIA2300 series vector with GFP fluorescent selection marker. The constructed recombinant vector was transformed into Agrobacterium K599 competent cells by freeze-thaw method, plated on LB solid medium containing 50 mg / L kanamycin, and cultured at 28℃ for 1-2 days. Single colonies were picked and inoculated into LB liquid medium containing the same antibiotic, and cultured at 28℃ and 180 rpm on a shaker until OD600 = 0.6-0.8. The cells were collected by centrifugation at 8000 rpm for 10 min, resuspended in infection solution (1 / 2 MS liquid medium + 150 μmol / L acetylsyl syringone + 0.02% Tween-20, pH 5.8), and the OD600 was adjusted to 0.6-0.8. The cells were then activated by static incubation at 28℃ in the dark for 30 min for later use.

[0010] Pretreatment and negative pressure infection: The pretreatment solution was 1 / 2 MS liquid medium + 100 μmol / L acetylsyringone + 0.02% Tween-20. The explants were soaked in the dark at 25℃ for 30 min. The pretreated explants were completely immersed in the infection solution and infected under a negative pressure of 0.05 MPa for 8-12 min. After the negative pressure was removed, the explants were allowed to stand at normal pressure in the dark for 5 min.

[0011] Co-culture and gradient sterilization: The co-culture medium was 1 / 2 MS solid medium + 150 μmol / L acetylsyl syringone + 30 g / L sucrose + 7.5 g / L agar, pH 5.8, and the co-culture conditions were 25℃ in the dark for 48 h. The gradient sterilization method was as follows: After co-culture, the explants were first rinsed 3 times with sterile water, then soaked in sterile water containing 300 mg / L termethin for 10 min, rinsed twice with sterile water, then soaked in sterile water containing 200 mg / L termethin for 5 min, and finally rinsed 3 times with sterile water and the surface moisture was absorbed with sterile filter paper.

[0012] Delayed culture and gradient resistance screening: The delayed culture medium was 1 / 2 MS solid medium + 300 mg / L termethin + 30 g / L sucrose + 7.5 g / L agar, pH 5.8, culture conditions were 25±1℃, light 16 h / d, light intensity 6000 lx, culture time 7 days; Gradient resistance screening was divided into three stages: First gradient (7 days): 1 / 2 MS solid medium containing 30 mg / L kanamycin + 300 mg / L termethin; Second gradient (14 days): 1 / 2 MS solid medium containing 20 mg / L kanamycin + 200 mg / L termethin; Third gradient (14 days): 1 / 2 MS solid medium containing 10 mg / L kanamycin + 100 mg / L termethin. Fresh medium was replaced every 7 days until hairy roots ≥2 cm in length grew from the explant wound.

[0013] Identification and root-strengthening culture of positive hairy roots: GFP fluorescence expression in hairy roots was observed using stereofluorescence microscopy. Hairy roots with uniform fluorescence signals were screened, and genomic DNA was extracted. PCR amplification and sequencing verification were performed using exogenous gene-specific primers and GFP tag primers to confirm positive transformation events. Single-clonal segments of positive hairy roots were excised and inoculated into root-strengthening medium (MS solid medium + 0.2 mg / L NAA + 0.1 mg / L 6-BA + 30 g / L sucrose + 7.5 g / L agar, pH 5.8), and cultured statically at 25±1℃ in the dark. Subculture was performed every 15 days to complete propagation and root strengthening.

[0014] Compared with the prior art, the beneficial effects of the present invention are: This invention uses semi-lignified root segments with axillary buds as explants. The wounds formed by the 45° oblique cut increase the infection sites of Agrobacterium. Combined with negative pressure assisted infection technology, the transformation efficiency of Agrobacterium is significantly improved. Experimental verification shows that the induction rate of hairy roots of wolfberry in this invention can reach more than 85%, and the positive transformation rate can reach more than 70%, which is far higher than the average level of 30-40% of the existing technology.

[0015] This invention employs a two-step screening system combining delayed culture and gradient resistance screening to gradually eliminate false-positive transformants and chimeras. Combined with hairy root monoclonal isolation culture and propagation, the resulting transgenic wolfberry root system exhibits excellent genetic stability. Verification has shown that the positive rate of T0 generation regenerated plants can reach over 90%, with no chimerism, and exogenous genes can be stably integrated and expressed within the transgenic hairy root system.

[0016] This invention demonstrates extremely high transformation efficiency for all major Chinese wolfberry varieties, including Ningqi No. 1, Ningqi No. 5, Ningqi No. 7, and Ningnongqi No. 9, with hairy root induction rates consistently above 80%. It breaks through the genotype limitation of existing transformation systems that are only effective for single varieties and can be adapted for large-scale wolfberry genetic transformation operations.

[0017] This invention establishes a standardized operating system for the entire process, requiring only 45 days from explant preparation to obtaining positive hairy roots, which is much shorter than the transformation cycle of more than 6 months in existing technologies, thus significantly reducing the time cost and operational difficulty of genetic transformation of wolfberry.

[0018] This invention is a universal goji berry genetic transformation system, which is not limited to the transformation of specific exogenous genes. It can be adapted to various plant binary expression vectors. It can not only be used for rapid functional verification of goji berry root-related genes, but also be widely applied in multiple fields such as improving goji berry stress resistance traits, regulating the synthesis of secondary metabolites, and creating new transgenic varieties. It provides a stable and efficient core technology platform for goji berry molecular breeding. Attached Figure Description

[0019] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 A flowchart illustrating the method for inducing transgenic roots of Lycium barbarum mediated by Agrobacterium rhizogenes.

[0020] Figure 2 A schematic diagram illustrating the process of preparing wolfberry explants, infection, and hairy root induction using Agrobacterium rhizogenes-mediated transgenic root induction of wolfberry.

[0021] Figure 3 The graph shows the GFP expression effect of positive transgenic hairy roots of Lycium barbarum induced by Agrobacterium rhizogenes under bright field and fluorescence.

[0022] Figure 4 The image shows the whole-plant fluorescence expression effect induced by the hairy roots of positive transgenic Lycium barbarum using Agrobacterium rhizogenes-mediated root induction method. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Please see Figure 1-4 The present invention provides the following technical solution: a method for inducing transgenic roots of Lycium barbarum mediated by Agrobacterium rhizogenes, comprising the following embodiments: Example 1: Induction of transgenic roots of Ningqi No. 1 with GFP reporter gene: S1. Preparation of sterile explants of Lycium barbarum: Mature seeds of Ningqi No. 1 were selected, surface-sterilized with 75% ethanol for 1 min, rinsed twice with sterile water, sterilized with 2% sodium hypochlorite solution for 7 min, rinsed 6 times with sterile water, and the surface moisture was absorbed with sterile filter paper. The seeds were then inoculated into 1 / 2 MS basic medium (30 g / L sucrose + 7.5 g / L agar, pH 5.8) and cultured for 14 days at 25±1℃, 16 h / d light, and 6000 lx light intensity to obtain sterile tissue culture seedlings with a height of 10-12 cm and 12-15 true leaves. Semi-lignified stem segments with 1-2 plump axillary buds and a length of 3-4 cm were cut from the base of the sterile seedlings. The lower end of the stem segment was cut at a 45° angle to form a root severance infection wound, and the upper end was cut horizontally, retaining 1 healthy true leaf to obtain the explants.

[0025] S2. Preparation of Infected Bacterial Culture: The pCAMBIA2300-GFP vector carrying the GFP fluorescent tag was transformed into Agrobacterium rhizogenes K599 competent cells by freeze-thaw method. The cells were plated on LB solid medium containing 50 mg / L kanamycin and cultured at 28°C for 2 days. Single colonies were picked and inoculated into LB liquid medium containing the same antibiotic. The cells were cultured at 28°C and 180 rpm on a shaker until OD600 = 0.7. The cells were collected by centrifugation at 8000 rpm for 10 min and resuspended in infection solution (1 / 2 MS liquid medium + 150 μmol / L acetylsyl syringone + 0.02% Tween-20, pH 5.8). The OD600 was adjusted to 0.7 and activated by static incubation at 28°C in the dark for 30 min for later use.

[0026] S3. Pretreatment and negative pressure infection: The explants were placed in the pretreatment solution (1 / 2 MS liquid medium + 100 μmol / L acetylsyl syringone + 0.02% Tween-20) and soaked at 25°C in the dark for 30 min. The pretreated explants were completely immersed in the infection solution and infected under a negative pressure of 0.05 MPa for 10 min. After the negative pressure was removed, the explants were allowed to stand at normal pressure in the dark for 5 min.

[0027] S4. Co-culture and gradient sterilization: After infection, the explants were inoculated into co-culture medium (1 / 2 MS solid medium + 150 μmol / L acetylsalicylic acid + 30 g / L sucrose + 7.5 g / L agar, pH 5.8) and cultured at 25°C in the dark for 48 h. After co-culture, the explants were rinsed 3 times with sterile water, then soaked in sterile water containing 300 mg / L termethin for 10 min, rinsed 2 times with sterile water, soaked in sterile water containing 200 mg / L termethin for 5 min, and finally rinsed 3 times with sterile water and dried with sterile filter paper.

[0028] S5. Delayed Culture and Gradual Resistance Screening: After sterilization, explants were inoculated into delayed culture medium (1 / 2 MS solid medium + 300 mg / L termethin + 30 g / L sucrose + 7.5 g / L agar, pH 5.8) and cultured for 7 days at 25±1℃, 16 h / d light, and 6000 lx light intensity. Subsequently, a gradient resistance screening was performed: the first gradient was inoculated into 1 / 2 MS solid medium containing 10 mg / L kanamycin + 300 mg / L termethin and cultured for 7 days; the second gradient was transferred to 1 / 2 MS solid medium containing 20 mg / L kanamycin + 200 mg / L termethin and cultured for 14 days; the third gradient was transferred to 1 / 2 MS solid medium containing 25 mg / L kanamycin + 100 mg / L termethin and cultured for 14 days, with fresh medium replaced every 7 days during the process.

[0029] S6. Identification of positive hairy roots and root-strengthening culture: After culture, the hairy root induction rate at the explant wound reached 89.2%; observation by stereofluorescence microscopy showed that 72.3% of the hairy roots showed a uniform green fluorescent signal; genomic DNA was extracted from the fluorescent positive hairy roots and PCR amplification was performed using GFP-specific primers. The sequencing results of the amplified products completely matched the GFP gene sequence, confirming the positive transformation event; single-clonal segments of the positive hairy roots were cut and inoculated into root-strengthening culture medium, cultured at 25±1℃ in the dark, and subcultured every 15 days. The hairy roots grew vigorously and the genetic traits were stable.

[0030] Example 2: Induction of transgenic roots of Ningqi 7 with PSY1 gene: In this embodiment, the target exogenous gene is the rate-limiting enzyme gene PSY1 for the synthesis of carotenoids in wolfberry, the recombinant vector is pCAMBIA2300-PSY1-GFP, the wolfberry variety used is Ningqi No. 7, and the remaining operation steps and culture medium formula are completely consistent with those in Example 1.

[0031] The experimental results showed that the hairy root induction rate of Ningqi No. 7 explants reached 86.7%, and the positive hairy root transformation rate reached 70.5%. The expression level of PSY1 gene in positive hairy roots was 4.2 times higher than that in wild-type roots, and the carotenoid content was 3.8 times higher. The adventitious bud differentiation rate reached 62.3%, and the positive rate of regenerated plants reached 89.7%. The exogenous gene can be stably integrated and expressed, which verifies the application effect of this invention in improving the quality traits of wolfberry.

[0032] Example 3: Induction of transgenic roots of Ningqi No. 5 with the DREB2A stress resistance gene: In this embodiment, the target exogenous gene is the Lycium barbarum stress resistance regulation gene DREB2A, the recombinant vector is pCAMBIA2300-DREB2A-GFP, the tested Lycium barbarum variety is Ningqi No. 5, and the remaining operation steps and culture medium formula are completely consistent with those in Example 1.

[0033] The experimental results showed that the hairy root induction rate of Ningqi No. 5 explants reached 85.1%, and the positive hairy root conversion rate reached 68.9%. Under 200 mmol / L NaCl salt stress treatment, the relative growth of positive hairy roots was 2.7 times higher than that of wild-type roots, SOD activity was 3.1 times higher, and malondialdehyde content was 45.2% lower, showing extremely strong salt tolerance. The positive rate of regenerated plants reached 88.2%, and the growth of T0 generation plants under salt stress was significantly better than that of wild-type plants, verifying the application value of this invention in improving the stress resistance traits of wolfberry.

[0034] 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 inducing transgenic roots of Lycium barbarum mediated by Agrobacterium rhizogenes, characterized in that, Includes the following steps: S1. Preparation of sterile explants of wolfberry: obtain sterile tissue culture seedlings of wolfberry, cut semi-lignified stem segments with axillary buds, and prepare explants with root severance wounds; S2. Preparation of infecting bacterial solution: Transform the plant binary expression vector carrying the target exogenous gene into Agrobacterium rhizogenes to prepare the activated infecting bacterial solution; S3. Pretreatment and negative pressure infection: The explants were pre-cultured in a pretreatment solution containing acetylsuccinone, and then infected with Agrobacterium using a negative pressure-assisted infection method. S4. Co-culture and gradient sterilization: The infected explants were placed in a co-culture medium and cultured in the dark. After completion, the Agrobacterium tumefaciens was sterilized by gradient immersion. S5. Delayed culture and gradient resistance screening: After sterilization, the explants were first subjected to antibiotic-free delayed culture, and then cultured in stages using resistance screening medium with increasing concentration gradients to induce the formation of positive hairy roots. S6. Identification of positive hairy roots and root-strengthening culture: Positive transgenic hairy roots were screened by fluorescent labeling and molecular biological identification, and then inoculated into root-strengthening culture medium to complete propagation and root strengthening.

2. The method for inducing transgenic roots of Lycium barbarum mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: The specific method for preparing aseptic explants of wolfberry in S1 is as follows: Select mature wolfberry seeds, disinfect them on the surface, and inoculate them into 1 / 2 MS basic medium. Culture them for 14 days at 25±1℃, light intensity 16h / d, and light intensity 6000lx to obtain aseptic tissue culture seedlings with a height of 10-12cm and 12-15 true leaves. Cut semi-lignified stem segments with 1-2 plump axillary buds and a length of 3-4cm from the base of the aseptic seedlings. Make a 45° oblique cut at the lower end of the stem segment to form a root severance infection wound, and make a horizontal cut at the upper end, retaining 1 healthy true leaf to obtain the explant.

3. The method for inducing transgenic roots of Lycium barbarum mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: The *Agrobacterium rhizogenes* in S2 is strain K599, and the plant binary expression vector is a pCAMBIA2300 series vector with a GFP fluorescent selection marker. The preparation method of the infection solution is as follows: a single colony of transformed *Agrobacterium* is inoculated into LB liquid medium containing 50 mg / L kanamycin, cultured at 28°C and 180 rpm on a shaker until OD600 = 0.6-0.8, centrifuged at 8000 rpm for 10 min to collect the cells, resuspended the cells in the infection solution and adjusted OD600 = 0.6-0.8, and activated by static incubation at 28°C in the dark for 30 min for later use. The formulation of the infection solution is: 1 / 2 MS liquid medium + 150 μmol / L acetylsyringone + 0.02% Tween-20, pH 5.

8.

4. The method for inducing transgenic roots of Lycium barbarum mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: The pretreatment solution in S3 is formulated as follows: 1 / 2 MS liquid culture medium + 100 μmol / L acetylsyringone + 0.02% Tween-20. The pretreatment conditions are: soaking at 25°C in the dark for 30 min. The specific parameters for the negative pressure assisted infection are: completely immersing the pretreated explants in the infection solution, infecting under a negative pressure of 0.05 MPa for 8-12 min, and then allowing them to stand at normal pressure in the dark for 5 min after the negative pressure is removed.

5. The method for inducing transgenic wolfberry roots mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: The S4 co-culture medium formulation is: 1 / 2 MS solid medium + 150 μmol / L acetylsalicylic acid + 30 g / L sucrose + 7.5 g / L agar, pH 5.8; the co-culture conditions are 25℃ in the dark for 48 h; the specific method of gradient sterilization is as follows: after co-culture, the explants are first rinsed 3 times with sterile water, then soaked in sterile water containing 300 mg / L termethin for 10 min, rinsed 2 times with sterile water, then soaked in sterile water containing 200 mg / L termethin for 5 min, and finally rinsed 3 times with sterile water and the surface moisture is absorbed with sterile filter paper.

6. The method for inducing transgenic roots of Lycium barbarum mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: The culture medium for delayed culture in S5 was: 1 / 2 MS solid medium + 300 mg / L termethin + 30 g / L sucrose + 7.5 g / L agar, pH 5.8, culture conditions were 25±1℃, light intensity 16 h / d, light intensity 6000 lx, and culture time 7 days; the gradient resistance screening was divided into three stages: First-tier screening: Explants after delayed culture were inoculated into 1 / 2 MS solid medium containing 30 mg / L kanamycin + 300 mg / L termethin and cultured for 7 days; Second-level screening: Transferred to 1 / 2 MS solid medium containing 20 mg / L kanamycin + 200 mg / L termethin, and cultured for 14 days; Third-tier screening: Transfer to 1 / 2 MS solid medium containing 10 mg / L kanamycin + 100 mg / L termethin, culture for 14 days, and replace with fresh medium every 7 days until hairy roots with a length ≥2 cm grow from the explant wound.

7. The method for inducing transgenic roots of Lycium barbarum mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: The molecular biological identification in S6 is PCR amplification identification, with primers being exogenous gene-specific primers and GFP tag primers; the root-strengthening culture medium formula is: MS solid medium + 0.2 mg / L NAA + 0.1 mg / L 6-BA + 30 g / L sucrose + 7.5 g / L agar, pH 5.8, culture conditions are 25±1℃, static culture in the dark, and subculture every 15 days.

8. The method for inducing transgenic roots of Lycium barbarum mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: The root-strengthening culture medium formula in S6 is: 1 / 2 MS solid medium + 0.2 mg / L IBA + 20 g / L sucrose + 7.5 g / L agar, pH 5.8; the genetic stability verification is performed by fluorescence observation and PCR sequencing on the T0 generation re-rooted system to confirm the stable integration and expression of exogenous genes in the whole genome of the plant.

9. A method for inducing transgenic roots of Lycium barbarum mediated by Agrobacterium rhizogenes according to any one of claims 1-8, characterized in that: The goji berries mentioned include the main cultivated goji berry varieties Ningqi No. 1, Ningqi No. 5, Ningqi No. 7, and Ningnongqi No. 9.

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