Construction method of Hainan camellia oleifera seedling transgenic hairy root system

Abstract

The invention belongs to the technical field of plant genetic engineering and biotechnology, and discloses a construction method of a Hainan camellia oleifera seedling transgenic hairy root system, which comprises the following steps: selecting a 3-month-old camellia oleifera seedling explant Haidao camellia oleifera No.4, pretreating, transferring a target plasmid into agrobacterium rhizogenes K599 by an agrobacterium-mediated method, and culturing to obtain the Hainan camellia oleifera seedling transgenic hairy root system. Preparing an infection solution, optimizing infection conditions, inducing hairy roots to generate, and finally verifying the transgenosis effect through fluorescence observation and molecular identification. According to the method, an infection liquid formula, infection conditions and culture conditions are optimized, the induction efficiency of the transgenic hairy roots is remarkably improved, technical support is provided for functional gene research and genetic improvement of Hainan camellia oleifera, and the method has important application value.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and biotechnology, and specifically relates to a method for constructing a transgenic hairy root system of Hainan camellia seedlings. Background Technology

[0002] Camellia oleifera ( Camellia Camellia spp. (family Theaceae) refers to the genus Camellia (family Theaceae) whose seeds have a high oil content and are valuable for cultivation. Camellia Camellia oil is a general term for edible vegetable oilseeds. It is rich in unsaturated acids and is a high-quality edible oil. In addition, camellia oil contains various active substances such as tea polyphenols, camelliaside, and squalene, which have good medicinal and health benefits.

[0003] The main cultivated species of Camellia oleifera in my country include common Camellia oleifera (Camellia oleifera) Camellia oleifera ), small-fruited tea oil ( Camellia meiocarpa ), Zhejiang Red Camellia ( Camellia chekiangoleosa ), Vietnamese camellia oil ( Camellia vietnamensis ) and Gaozhou camellia oil ( Camellia gauchowensis ), etc. Among them, Vietnamese camellia oil ( C. vietnamensis Camellia oleifera (Zanthoxylum bungeanum) is the main cultivated camellia species in Hainan Province. It has long adapted to the unique tropical monsoon climate, forming a "geographical minor" due to the island's unique geographical location. It possesses significant potential for upgrading the camellia oil industry and developing functional plant oils, and is particularly suitable for the development and utilization of high-efficiency oil crops. It is necessary to strengthen research in areas such as improved variety breeding, developmental biology, cytology, and genetics of Camellia oleifera in Hainan Province to promote the sustainable utilization of Camellia oleifera resources in tropical regions.

[0004] In recent years, research on camellia oleifera has mainly focused on optimizing oil extraction processes, fatty acid composition analysis techniques, and external cultivation management measures such as pruning and shaping of field plants. Compared to other major herbaceous oil crops such as rapeseed and peanuts, camellia oleifera, as a woody oil crop, has relatively weak foundations in breeding superior varieties and basic physiological and biochemical research during key growth stages due to its perennial nature, resulting in slower progress in improving the quality and efficiency of the industry. Suitable planting areas for camellia oleifera generally suffer from low efficiency in breeding superior varieties and insufficient effectiveness in improving planting quality. Genetic engineering, the core of modern biotechnology, combined with traditional breeding techniques for genetic improvement of camellia oleifera trees, can effectively accelerate the efficiency of breeding superior varieties.

[0005] However, an efficient and stable genetic transformation system for Camellia oleifera has not yet been established. This situation has become a major bottleneck for its genome function analysis and verification, as well as the delivery of gene editing tools. It has also limited the discovery and utilization of functional genes in Camellia oleifera to some extent, thus affecting the further application of genetic engineering technology in the genetic improvement of Camellia oleifera. Summary of the Invention

[0006] To achieve the above-mentioned purposes of the application, the application adopts the following technical solutions. A method for constructing a transgenic hairy root system of Hainan Camellia sinensis seedlings, comprising the following steps: (1) Preparation of Camellia sinensis seedling explants: Select 3-month-old Hainan Camellia sinensis seedlings as starting test materials; wash the root culture medium of the Camellia sinensis seedlings, absorb the water on the surface of the plants with absorbent paper, and cut each leaf of the Camellia sinensis seedling to leave only 1 / 3 of the leaf with sterile scissors, cut off the lower part of the main root and the excess lateral roots, leaving the main root about 2-3 cm long, and place the treated Camellia sinensis seedlings in a well-ventilated, cool place to dry the roots thoroughly, and then use them as explants for Agrobacterium infection; (2) Construction of Agrobacterium tumefaciens engineering bacteria containing target genes and: Transfer the plasmid pCXSN-EGFP containing the target gene into the competent cells of Agrobacterium tumefaciens K599, and obtain the engineering bacteria containing the target gene through PCR identification; (3) Preparation of infection solution: Activate and expand the culture of the engineering bacteria; after the culture is completed, centrifuge and collect the bacterial cells, resuspend the bacterial cells with the same volume of infiltration solution without hormones, and adjust the concentration of the bacterial solution to OD 600 = 0.9 to obtain the infection solution; wherein the composition of the infiltration solution is: 1 / 2MS+30 g / L sucrose+CW 5 %+AS 20 mg / L; (4) Infection of explants and induction culture of transgenic hairy roots: Use a sterile dissecting needle to make 1-3 wounds on the root stem of the explants and immerse them in the infection solution, and use the soaking and shaking method to infect for 30 minutes; take out the infected explants, absorb the bacterial solution on the surface of the explants with sterile filter paper, and transplant them into sterile vermiculite for induction culture for 35-50 days; wherein the culture conditions of the sterile vermiculite are: temperature 24±2℃, 16 hours light / 8 hours dark cycle; (5) Fluorescence detection and molecular identification of transgenic hairy roots: After 50 days of culture, the hairy roots induced at the base of the Camellia sinensis seedling stems are observed for fluorescence and molecular identification.

[0007] Further, the Hainan Camellia sinensis seedlings are 3-month-old seedlings induced from the germination of 'Haidao Camellia No. 4' Camellia sinensis embryos.

[0008] Further, the plasmid pCXSN-EGFP containing the target gene is transformed into the Agrobacterium rhizogenes K599 competent cell, including the following steps: the Agrobacterium rhizogenes competent cell stored at -80°C is placed in ice water for 5 minutes, then 500 ng of the target plasmid DNA is added and mixed by blowing, and then the alternating treatment of ice water for 5 minutes, liquid nitrogen for 5 minutes, 37°C water bath for 5 minutes, and ice water for 5 minutes is sequentially performed; after the ice water treatment is completed, 400 μL of LB liquid medium is added, and the culture is shaken at 28°C and 200 rpm for 1 hour; the bacteria are collected by centrifugation at 5000 rpm for 1 minute, the supernatant is discarded, and about 80 μL of the resuspended bacteria is retained; the bacterial solution is spread on the LB plate containing the corresponding antibiotic, and then the plate is cultured at 28°C for 2 days; after the colonies grow, a single colony is picked for PCR identification, and the positive single colony is the Agrobacterium rhizogenes engineering bacteria containing the target gene; the engineering bacteria is transferred to the LB liquid medium containing the corresponding antibiotic, and the culture is shaken at 28°C and 200 rpm for 12 hours; 750 μL of the bacterial solution is taken into a 2 mL centrifuge tube, and an equal volume of 50% glycerol is added, and then the mixture is stored at -80°C for standby.

[0009] Further, the activation and expansion culture of the engineering bacteria includes the following steps: the engineering bacteria is streaked on the LB plate containing kanamycin and streptomycin, and then the plate is cultured at 28°C for 2 days; a single colony is selected and inoculated into the LB liquid medium containing the same antibiotic, and then the culture is shaken for 12 hours; 1 mL of the bacterial solution is taken from the culture at a ratio of 1:100, and then the mixture is added into 100 mL of the LB liquid medium, and then the mixture is cultured under the same conditions for about 8 hours.

[0010] Further, the centrifugation is performed by a high-speed centrifuge at 4°C and 5000 rpm for 10 min.

[0011] Further, the fluorescence observation is performed by using an excitation light source to observe the fluorescence of the transgenic hairy roots.

[0012] Further, the molecular identification includes the following steps: 0.5 g of fresh hairy roots is taken, ground into powder after being added with liquid nitrogen, and then the genomic DNA of the hairy roots is extracted by using the CTAB method; the extracted genomic DNA of the hairy roots is used as a template, and then the specific primers for the Agrobacterium rhizogenes Ri plasmid rolB gene, the pCXSN-EGFP plasmid Hyg gene, and the pCXSN-EGFP plasmid EGFP gene are used for PCR amplification detection; The nucleotide sequence of the specific primer rolB -F is shown in SEQ ID NO. 1.rolB the nucleotide sequence of -R is shown as SEQ ID NO. 2, the specific primer Hyg the nucleotide sequence of -F is shown as SEQ ID NO. 3, the specific primer Hyg the nucleotide sequence of -R is shown as SEQ ID NO. 4, the specific primer EGFP the nucleotide sequence of -F is shown as SEQ ID NO. 5, the specific primer EGFP the nucleotide sequence of -R is shown as SEQ ID NO. 6.

[0013] Advantages of the present application: The present application establishes a high-efficiency transgenic hairy root system suitable for Camellia hainanensis seedlings by optimizing the pretreatment of explants, Agrobacterium transformation system and infection conditions. The system uses the basal part of 3-month-old Camellia hainanensis variety 'Haidaoyoucha No. 4' seedling as the explant, significantly improves the induction efficiency of transgenic hairy roots by pretreating the explants and introducing specific infection liquid formula and culture conditions. In addition, the present application combines fluorescence observation and molecular identification technology to ensure the accuracy and reliability of transgenic hairy roots. This technical system lays a foundation for the research and genetic improvement of functional genes of Camellia hainanensis, and provides technical support for the sustainable use and efficient development of Camellia hainanensis resources in the future. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 It is the process photo of activation and expansion culture of Agrobacterium K599 of the present application (A. Agrobacterium plate; B. Agrobacterium small shake liquid; C. Agrobacterium large shake liquid; D. Infection liquid).

[0015] Figure 2 It is the infection process photo of Camellia seedlings of the present application (A. Wild type Camellia seedling; B. Treated explant; C. Explant infection; D. Explant culture E. 35 d hairy root induction F. 60 d rooting condition).

[0016] Figure 3 It is the fluorescence detection result photo of hairy roots of the present application (A. Uninfected Camellia seedling bright field; B-C. Infected Camellia seedling bright field; D. Uninfected Camellia fluorescence field E-F. Infected Camellia seedling fluorescence field).

[0017] Figure 4 It is the PCR detection result photo of hairy roots of the present application (M: marker; T1-T5 are Camellia positive hairy root samples; WT: wild type root sample; P: bacterial liquid sample). DETAILED DESCRIPTION

[0018] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments. Example

[0019] 1. Materials and Methods 1.1 Plant materials The camellia oleifera material used in this invention comes from the local Hainan camellia oleifera seedling 'Haida Camellia oleifera No. 4'. After the camellia oleifera seed embryos were induced to germinate, the 3-month-old seedlings were used as the starting experimental material.

[0020] 1.2 Test strains The strain used in this invention comprises *Agrobacterium rhizogenes* K599, purchased from Shanghai Weidi Biotechnology Co., Ltd. The plasmid containing the target gene used in this invention is the pCXSN-EGFP plasmid, which carries the enhanced green fluorescent protein gene (…). EGFP) .

[0021] 1.3 Activation of Agrobacterium rhizogenes K599 and preparation of infection solution 1.3.1 Transformation and identification of Agrobacterium rhizogenes K599 The plasmid containing the target gene was transformed into *Agrobacterium rhizogenes*: *Agrobacterium rhizogenes* competent cells stored at -80℃ were incubated on ice for 5 minutes, then 500 ng of plasmid DNA was added and mixed by pipetting. This process was repeated alternately with ice incubation for 5 minutes, liquid nitrogen incubation for 5 minutes, water incubation at 37℃ for 5 minutes, and ice incubation for 5 minutes. After the ice incubation, 400 μL of LB liquid medium was added, and the culture was incubated at 28℃ and 200 rpm for 1 hour with shaking. The cells were collected by centrifugation at 5000 rpm for 1 minute, the supernatant was discarded, and approximately 80 μL of the resuspended cells was collected. The bacterial suspension was spread onto LB agar plates containing the corresponding antibiotics, inverted, and incubated at 28℃ for 2 days. After colonies grew, single colonies were picked for PCR identification. Positive single colonies were identified as engineered *Agrobacterium rhizogenes* containing the target gene. The engineered cells were then transferred to LB liquid medium containing the corresponding antibiotics and incubated at 28℃ and 200 rpm for 12 hours with shaking. 750 μL of the culture was then collected. The bacterial culture was transferred to a 2 mL centrifuge tube, and an equal volume of 50% glycerol was added. The tube was then stored at -80°C for later use.

[0022] 1.3.2 Preparation of Infection Solution Will carry EGFPAgrobacterium strain K599 was streaked onto LB agar plates containing kanamycin and streptomycin, and incubated upside down at 28 °C for 2 days. Single colonies were then inoculated into LB liquid medium containing the same antibiotics and cultured with shaking for 12 h. Next, 1 mL of the bacterial suspension was diluted 1:100 and added to 100 mL of liquid medium, and cultured again under the same conditions with shaking for approximately 8 h. After incubation, the bacterial cells were collected by centrifugation at 4 °C and 5000 rpm for 10 min. The cells were then resuspended in an equal volume of hormone-free infiltration solution. The bacteria were then resuspended in an infiltration solution (1 / 2 MS + 30 g / L sucrose + 5% CW + 20 mg / L AS), and the bacterial concentration was adjusted to OD. 600 The concentrations were 0.6, 0.9, and 1.2, respectively, to obtain the inoculum.

[0023] Table 1. Culture media and components used for Agrobacterium infection of explants

[0024] 1.4 Preparation of explants Three-month-old Camellia oleifera seedlings from Hainan were selected as the initial experimental material. The culture medium at the roots of the Camellia oleifera seedlings was washed clean, and the surface moisture of the plants was dried with absorbent paper. Each leaf of the Camellia oleifera seedling was cut off to only 1 / 3 of the leaf with sterile scissors. The lower part of the main root and the excess lateral roots were cut off, leaving the main root about 2-3 cm long. After the treated Camellia oleifera seedlings were placed in a ventilated and shady place to dry the roots thoroughly, they were used as explants for Agrobacterium tumefaciens infection. Using a sterile dissecting needle, 1-3 wounds were made on the rhizome of the prepared explant. The explant was then placed in an invasion dye solution, covering the wounds. Infection methods included: injection, dipping, and soaking with shaking. After removal, the bacterial solution on the surface of the explant was blotted dry with sterile filter paper. The explant was then transplanted into sterile vermiculite at a temperature of 24±2°C, with a 16-h light / 8-h dark cycle. After induction for about 35-50 days, the induced adventitious roots could be observed and detected for fluorescence.

[0025] To find the most suitable infection conditions, the bacterial concentration, infection mode, and infection duration were analyzed using L9(3) assays. 3 Orthogonal experiment, bacterial concentration is OD 600 =0.6, 0.9, 1.2; infection time set to 10 min, 20 min, 30 min.

[0026] 1.5 Data Processing Data were processed using Microsoft Excel 2010 and SPSS 21.0, and the results were subjected to one-way ANOVA. The survival rate = (survival plants / total number of plants inoculated) x 100, the hairy root induction rate = (hairy root plants / survival plants) x 100, and the hairy root transformation rate = (green fluorescent hairy root plants / hairy root plants) x 100.

[0027] 1.6 DNA extraction and molecular identification of transgenic hairy roots 0.5 g of fresh hairy roots were placed in a mortar and ground to a powder after the addition of liquid nitrogen. The SteadyPure Plant Genomic DNA Extraction Kit (CTAB method) from Aikong Biological Co., Ltd. was used to extract the hairy root DNA, which was used as a template for subsequent gene identification.

[0028] Oligo 7 software was used to design specific primers for the rolB gene of Agrobacterium rhizogenes Ri plasmid B The specific primers for the rolB gene of Agrobacterium rhizogenes Ri plasmid B -F / R (SEQ ID NO. 1 / SEQ ID NO. 2), specific primers for the Hyg gene on the pCXSN-EGFP plasmid Hyg -F / R (SEQ ID NO. 3 / SEQ ID NO. 4), and specific primers for the EGFP gene on the pCXSN-EGFP plasmid EGFP -F / R (SEQ ID NO. 5 / SEQ ID NO. 6) were synthesized by Qianke Biological Co., Ltd. The extracted hairy root genomic DNA was used as a template, and the target gene was amplified using 2 x Phanta Max Master Mix. The reaction system was as follows: 1 μL DNA, 5 μL 2 x Phanta Max Master Mix (Nanjing Novozyme Biological Co., Ltd.), 0.8 μL forward primer, 0.8 μL reverse primer, and 2.4 μL ddH2O. The mixed reaction system was amplified using a PCR instrument, and the PCR reaction program was as follows: 95℃ for 3 min, 33 cycles of 95℃ for 15 s, 56℃ for 15 s, 72℃ for 25 s, 72℃ for 5 min, and 4℃ for ∞.

[0029] 2、Results analysis 2.1 Induction of hairy roots by inoculation of explants The infected explants were transplanted into sterile vermiculite, and the hairy roots of the leaves began to grow about 35-60 days after transformation. During the rooting process, callus-like objects first formed at the wound, and then hairy roots began to germinate. After six weeks of culture, the hairy roots gradually exhibited vigorous growth, resulting in transgenic hairy roots and wild-type stem and leaf composite plants.

[0030] 2.2 Influence of different infection conditions on transformation efficiency of hairy roots When the Camellia oleifera plant hairy roots grew, they were observed under an excitation light source and counted. According to the orthogonal test analysis of the influence of different treatments on the transformation efficiency of Camellia oleifera hairy roots, the primary and secondary relationships of the influence of the three factors on the transformation efficiency were as follows: bacteria liquid concentration > infection method > infection time, indicating that there were differences in the influence of different factors on the transformation efficiency. According to the orthogonal test table L9(3 3 ) factor level, the transformation efficiency of No. 6 was the highest, and the optimal combination was bacteria liquid concentration OD 600 = 0.9, infection by soaking and shaking for 30 min, and the highest transformation efficiency was 22.22% (Table 2).

[0031] Table 2 Influence of different infection treatments on transformation efficiency of Camellia oleifera hairy roots

[0032] Note: K1, K2, K3 refer to the comprehensive effect value of the test index under different levels of a factor, that is, the sum (total value) of the corresponding test results; k1, k2, k3 represent the number of times that the large K appears at each level, that is, the average value; R value is the range, the maximum minus the minimum in k1-3, and the larger the R value, the greater the influence.

[0033] 2.3 Fluorescence detection of marker protein EGFP After 50 days of infection, abundant hairy roots were induced at the base of the Camellia oleifera seedlings. The roots grew radially and had no obvious geotropism, which was consistent with the morphological response caused by overexpression of rol genes, indicating that the T-DNA of the Ri plasmid may have been successfully transferred to the root somatic cells of Camellia oleifera and induced cell dedifferentiation and redifferentiation. The transgenic hairy roots were observed for fluorescence using a dual-wavelength portable fluorescent protein excitation light source (LUYOR-3415RG), and the resistant calli, hairy roots, or plant organs of the transgenic Camellia oleifera obtained after resistance screening emitted green fluorescence under the fluorescent field, proving that the Camellia oleifera leaf transgenic hairy root system was successfully constructed. EGFP

[0034] 2.4 Molecular identification of Camellia oleifera transgenic hairy roots ​The newly born hairy roots were observed under excitation light to prove that the exogenous gene was successfully transformed. The analysis of rooting efficiency by optimization for the best level combination showed that the highest induction rate of adventitious roots was 80.07%, and the highest transformation efficiency of hairy roots was 45.26% (Table 3).

[0035] Table 3 Data statistics of adventitious root induction rate and hairy root transformation efficiency of Camellia oleifera plants after 50 days of infection

[0036] The Camellia oleifera treated by Agrobacterium rhizogenes K599 was infected for 50 days, and the K599 Agrobacterium liquid of EGFP gene plasmid was used as a positive control, and the wild type Camellia oleifera leaf hairy root DNA was used as a negative control. The electrophoresis results of PCR amplification products are shown in Figure 4 As shown in the table, samples 1-5 showed bands, indicating that Agrobacterium rhizogenes Ri plasmid rolB gene, Hyg and EGFP genes on the pCXSN-EGFP plasmid were detected. The bands of rolB, Hyg and EGFP genes were amplified, indicating that these genes had been successfully integrated into the Camellia oleifera hairy root genome, which was a transgenic hairy root.

Claims

1. A method for constructing a transgenic hairy root system of Camellia koushuensis seedlings, characterized by, Comprising the following steps: (1) Preparation of Camellia oleifera seedling explants: Select 3-month-old Hainan Camellia oleifera seedlings as starting test materials; wash the root culture medium of the Camellia oleifera seedlings, dry the surface water of the plants with absorbent paper, cut each leaf of the Camellia oleifera seedlings to leave only 1 / 3 of the leaf with sterile scissors, cut off the lower part of the main root and the excess lateral roots, leaving the main root about 2-3 cm long, and then place the treated Camellia oleifera seedlings in a ventilated and cool place to dry the roots thoroughly before using them as explants for Agrobacterium infection; (2) Construction of Agrobacterium tumefaciens engineering bacteria containing the target gene and: Transfer the plasmid pCXSN-EGFP containing the target gene into the competent cells of Agrobacterium tumefaciens K599, and identify by PCR to obtain engineering bacteria containing the target gene; (3) Preparation of infection solution: The engineering bacteria are activated and cultured in large scale; after the culture is completed, the bacteria are collected by centrifugation, the bacteria are resuspended with the same volume of infiltration solution without hormones, and the concentration of the bacterial solution is adjusted to OD 600 = 0.9 to obtain an infection solution; wherein the infiltration solution comprises: 1 / 2MS+30 g / L sucrose+CW 5%+AS 20 mg / L; (4) Infection of explants and induction of transgenic hairy roots: Use a sterile dissecting needle to make 1-3 wounds on the root stem of the explants and immerse them in the infection solution by soaking and shaking for 30 minutes; remove the infected explants, dry the surface bacterial solution with sterile filter paper, and transplant them into sterile vermiculite for induction and culture for 35-50 days; wherein the culture conditions of the sterile vermiculite are: temperature 24±2℃, 16 hours light / 8 hours dark cycle; (5) Fluorescence detection and molecular identification of transgenic hairy roots: After 50 days of culture, observe and identify the fluorescence of the hairy roots induced at the base of the Camellia oleifera seedling stems.

2. The method of claim 1, wherein, The Hainan Camellia oleifera seedlings are 3-month-old seedlings induced from the germination of 'Haidao Camellia 4' Camellia oleifera seeds.

3. The method of claim 1, wherein, The plasmid pCXSN-EGFP containing the target gene is transferred into the competent cells of Agrobacterium tumefaciens K599, comprising the following steps: take the competent cells of Agrobacterium tumefaciens stored at -80℃, ice bath for 5 minutes, then add 500 ng of target plasmid DNA and mix well by blowing, and then perform alternating treatment of ice bath for 5 minutes, liquid nitrogen for 5 minutes, 37℃ water bath for 5 minutes, and ice bath for 5 minutes in sequence; after ice bath, add 400 μL of LB liquid medium, and culture at 28℃, 200 rpm for 1 hour; centrifuge at 5000 rpm for 1 minute to collect the bacteria, discard the supernatant, and retain about 80 μL of resuspended bacterial bodies; spread the bacterial solution on LB plates containing the corresponding antibiotics, invert and culture at 28℃ for 2 days; after the colonies grow, pick single colonies for PCR identification, and the positive single colonies are the Agrobacterium tumefaciens engineering bacteria containing the target gene; transfer the engineering bacteria to LB liquid medium containing the corresponding antibiotics, and culture at 28℃, 200 rpm for 12 h; take 750 μL of bacterial solution into a 2 mL centrifuge tube, add an equal volume of 50% glycerol, and store at -80℃ for future use.

4. The method of claim 1, wherein, The activation and expansion culture of the engineering bacteria includes the following steps: the engineering bacteria are streaked on LB plate containing kanamycin and streptomycin, and are cultured at 28℃ for 2 days; a single colony is selected and inoculated into LB liquid medium containing the same antibiotics, and is shaken and oscillated for 12 hours; 1 mL of the bacterial solution is taken out at a ratio of 1:100, and is added into 100 mL of LB liquid medium, and is oscillated under the same condition for about 8 hours.

5. The method of claim 1, wherein, The centrifugation is performed by a high-speed centrifuge at 4℃ and 5000 rpm for 10 min.

6. The method of claim 1, wherein, The fluorescence observation is that the fluorescence observation of the transgenic hairy roots is performed by using an excitation light source.

7. The method of claim 1, wherein, The molecular identification comprises the following steps: taking 0.5 g of fresh hairy roots, grinding into powder after adding liquid nitrogen, and using CTAB method to extract the genomic DNA of the hairy roots; using the extracted hairy root genomic DNA as a template, respectively using specific primers rolB-F / R, rolC-F / R, rolD-F / R, rolE-F / R and rolG-F / R for the Agrobacterium rhizogenes Ri plasmid rolB genes to perform PCR amplification detection B Hyg genes on the pCXSN-EGFP plasmid Hyg EGFP genes on the pCXSN-EGFP plasmid EGFP genes on the pCXSN-EGFP plasmid​​ The nucleotide sequence of the specific primer -F is shown as SEQ ID NO. 1, and the nucleotide sequence of the specific primer -R is shown as SEQ ID NO.

2. rolB The nucleotide sequence of the specific primer -F is shown as SEQ ID NO. 1, and the nucleotide sequence of the specific primer -R is shown as SEQ ID NO.

2. rolB The nucleotide sequence of the specific primer -F is shown as SEQ ID NO. 1, and the nucleotide sequence of the specific primer -R is shown as SEQ ID NO.

2. Hyg The nucleotide sequence of the specific primer -F is shown as SEQ ID NO. 1, and the nucleotide sequence of the specific primer -R is shown as SEQ ID NO.

2. Hyg The nucleotide sequence of the specific primer -F is shown as SEQ ID NO. 1, and the nucleotide sequence of the specific primer -R is shown as SEQ ID NO.

2. EGFP The nucleotide sequence of the specific primer -F is shown as SEQ ID NO. 1, and the nucleotide sequence of the specific primer -R is shown as SEQ ID NO.

2. EGFP The nucleotide sequence of the specific primer -F is shown as SEQ ID NO. 1, and the nucleotide sequence of the specific primer -R is shown as SEQ ID NO. 2.

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