Establishment method and application of agrobacterium rhizogenes mediated woody plant non-genotype dependent root tiller regeneration transformation system

Abstract

The invention discloses an establishment method and application of an agrobacterium rhizogenes mediated woody plant non-genotype dependent root tiller regeneration transformation system, and belongs to the technical field of plant transgenosis. In order to solve the technical problem that the molecular breeding process of poplar is restricted due to lack of a poplar efficient genetic transformation system, a set of efficient and universal poplar genetic transformation system is established, specifically, a fluorescent labeled gene (EGFP / mCherry) is innovatively adopted to replace a traditional antibiotic screening marker, the screening period is shortened to be within 2 weeks from conventional 4-6 weeks, and the screening efficiency is greatly improved. The screening efficiency is obviously improved; meanwhile, a traditional multi-step transformation process is simplified into a single rooting culture medium and a bud induction culture medium by systematically optimizing a culture medium formula, so that the operation process is simplified, and the experiment cost is reduced. According to the method, data reference and technical support are provided for establishment of an efficient genetic transformation and gene editing system of all woody plants with sprout tillering capacity.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic transformation, and specifically relates to a method for establishing a genotype-independent root sucker regeneration transformation system mediated by Agrobacterium rhizogenes in woody plants and its application. Background Art

[0002] Populus spp., as fast-growing tree species widely distributed globally, have important strategic value in fields such as wood production, ecological restoration, and bioenergy development. However, genetic improvement of Populus faces many challenges, and the lack of an efficient genetic transformation system is one of the main technical bottlenecks restricting its molecular breeding process. Traditional genetic transformation techniques mainly rely on Agrobacterium tumefaciens-mediated method and PEG-mediated method. However, these methods generally have problems such as low transformation efficiency (usually <30%), significant genotype dependence, and high chimerism ratio. Especially for woody plants, their complex secondary metabolites and cell wall structures further reduce the delivery efficiency of foreign genes. In recent years, the emergence of CRISPR / Cas9 genome editing technology has provided a powerful tool for plant genetic improvement, but its application in Populus is still limited by an efficient genetic transformation system.

[0003] The genetic transformation system mediated by Agrobacterium rhizogenes provides an innovative solution to the above problems. The T-DNA region carried by the Ri plasmid of this strain contains root induction genes (rolA / B / C) and auxin synthesis genes (aux1 / 2), which can efficiently induce the differentiation of hairy roots without exogenous hormone regulation. The hairy root system has the characteristic of single-cell origin, which can significantly reduce the incidence of chimeras. At the same time, its fast growth characteristic is conducive to shortening the genetic transformation cycle. However, existing research is mostly limited to herbaceous plants, and there are still defects such as the lack of transformation methods for native tree species, low transformation efficiency, and the lack of gene editing systems in the application of woody plants, especially Populus. Summary of the Invention

[0004] In order to solve the technical problem that the lack of an efficient genetic transformation system in Populus restricts its molecular breeding process, the present invention has established a set of efficient and universal Populus genetic transformation systems. Specifically, by innovatively using fluorescent marker genes (EGFP / mCherry) to replace traditional antibiotic selection markers, the screening period is shortened from the conventional 4 - 6 weeks to within 2 weeks, significantly improving the screening efficiency; at the same time, by systematically optimizing the culture medium formula, the traditional multi-step transformation process is simplified into a single rooting culture medium and a bud induction culture medium, simplifying the operation process and reducing the experimental cost.

[0005] To solve the above technical problems and achieve corresponding technical effects, the present invention specifically provides the following technical solutions:

[0006] The first object of the present invention is to provide a method for establishing an Agrobacterium rhizogenes-mediated genotype-independent root suckering regeneration transformation system for woody plants. The establishment method includes the following steps:

[0007] S1. Transfer the expression vector containing the target gene into Agrobacterium rhizogenes to obtain Agrobacterium rhizogenes containing the target gene. The expression vector containing the target gene is based on the pCAMBIA1300 vector as the backbone, removing the resistance gene, and inserting the EGFP or mCherry fluorescent marker gene under the regulation of the 35S promoter.

[0008] S2. Cut the apical bud of the poplar tissue culture seedling, immerse the cut part in the Agrobacterium rhizogenes bacterial solution for infection, and insert it into the co-culture medium for co-culture after infection. After the co-culture is completed, wash it 3-5 times with sterile water containing Cef, transfer the washed stem segment to the WPM basal medium containing Cef, and after culturing for 30 days, a large number of white roots, namely hairy roots, will be formed.

[0009] S3. Use a stereoscopic fluorescence microscope to detect the expression of the EGFP or mCherry fluorescent marker gene in the hairy roots obtained in S2.

[0010] S4. Transfer the transgenic hairy roots aseptically to the shoot induction medium and culture for 34-56 days to obtain young shoots. Select the vigorously growing young shoots and transfer them to the rooting medium, and continue to culture for 2-3 weeks. After the roots are fully developed, obtain complete transgenic plants. The composition of the shoot induction medium is 2.41 g / L WPM, 25 g / L sucrose, 0.5 g / L MES, 0.02 mg / L TDZ, 6 g / L agar, and the balance is water, pH = 5.8. After sterilization, add 200 mg / L Cef.

[0011] In one embodiment of the present invention, the Agrobacterium rhizogenes in S1 is K599.

[0012] In one embodiment of the present invention, the poplar varieties in S2 include Populus trichocarpa, Populus ussuriensis, Populus simonii × Populus nigra, Nanlin 895 Poplar, 84K Poplar, Populus alba var. pyramidalis, Populus davidiana × Populus bolleana, and Populus alba var. berolinensis.

[0013] In one embodiment of the present invention, the OD of the Agrobacterium rhizogenes bacterial solution in S2 600nm is 0.6.

[0014] In one embodiment of the present invention, the composition of the co-culture medium in S2 is 2.41 g / L WPM, 25 g / L sucrose, 0.5 g / L MES, 6 g / L agar, with the balance being water, pH = 5.8, and 100 μM As is added after sterilization.

[0015] In one embodiment of the present invention, the conditions for co-culture in S2 are 25 ± 2 °C for 2 days of co-culture.

[0016] In one embodiment of the present invention, the concentration of Cef in the WPM basal medium in S2 is 400 mg / L, and the corresponding culture conditions for the WPM basal medium are 16 h of light, 8 h of darkness, a temperature of 25 ± 2 °C, and a relative humidity of 50 - 60%.

[0017] In one embodiment of the present invention, the specific steps of S3 are as follows: Wash the hairy roots with sterile water and perform fluorescence microscopy observation. The fluorescence detection parameters are set as follows: The excitation wavelength of mCherry is 587 nm, and the emission wavelength is 610 nm; the excitation wavelength of EGFP is 488 nm, and the emission wavelength is 510 nm. By observing the fluorescence signal, it is judged whether the foreign vector has been transferred into the hairy roots. The roots that emit red fluorescence at an excitation wavelength of 587 nm or green fluorescence at an excitation wavelength of 488 nm are the transgenic root systems.

[0018] In one embodiment of the present invention, the composition of the rooting medium in S4 is 2.41 g / L WPM, 25 g / L sucrose, 0.5 g / L MES, 6 g / L agar, with the balance being water, pH = 5.8, and 200 mg / L Cef is added after sterilization.

[0019] The second object of the present invention is to provide the application of the above establishment method in the breeding of woody plants.

[0020] The beneficial effects of the present invention:

[0021] The present invention has established a set of efficient and universal poplar genetic transformation systems. By innovatively using fluorescent marker genes (EGFP / mCherry) to replace traditional antibiotic screening markers, the screening period is shortened from the conventional 4 - 6 weeks to within 2 weeks, significantly improving the screening efficiency. At the same time, by systematically optimizing the medium formula, the traditional multi-step transformation process is simplified into a single rooting medium and a bud induction medium, simplifying the operation process and reducing the experimental cost. The present invention will provide data reference and technical support for the establishment of efficient genetic transformation and gene editing systems for all woody plants with the ability of sprouting.

[0022] The present invention has successfully optimized the Agrobacterium rhizogenes-mediated genetic transformation system of poplar. Innovatively, a fluorescent marker gene is used for screening, eliminating the need to rely on traditional resistance screening methods. This system can achieve efficient genetic transformation using only one type of culture medium, demonstrating remarkable universality. Experimental data shows that the average transgenic efficiency of this system exceeds 60%, and the gene editing efficiency reaches 39.2%. This highly efficient and stable genetic transformation system provides a reliable technical platform for the creation of new poplar germplasm resources, significantly promoting the progress of poplar molecular breeding research. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram for the construction of 1step Cas9 ATU6 EGFP and 1step Cas9 ATU6 mCherry vectors;

[0024] Figure 2 Map of the 1step Cas9 ATU6 EGFP vector;

[0025] Figure 3 Map of the 1step Cas9 ATU6 mCherry vector;

[0026] Figure 4 Figure showing the production of hairy adventitious roots in Populus simonii × P. nigra explants infected with Agrobacterium rhizogenes;

[0027] Figure 5 Result figure for the detection of transgenic positive hairy roots in Populus simonii × P. nigra;

[0028] Figure 6 Figure showing the induction process of root suckering adventitious buds in Populus simonii × P. nigra;

[0029] Figure 7 Result figure for the optimization of the formula of the bud induction medium;

[0030] Figure 8 Effect figure for the knockout of the PDS gene in 8 poplar germplasms using the transformation system provided by the present invention;

[0031] Figure 9 Figure showing three mutation patterns of PDS gene editing induced by CRISPR / Cas9; DETAILED DESCRIPTION OF THE INVENTION

[0032] The following further elaborates on the present invention in conjunction with specific embodiments and the accompanying drawings. The following embodiments facilitate a better understanding of the present invention but do not limit the present invention. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The drugs, reagents, and materials used in the following embodiments can all be obtained through commercial channels unless otherwise specified.

[0033] The plant materials involved in the present invention are as follows:

[0034] Eight poplar germplasms, such as Populus trichocarpa, Populus ussuriensis, Populus simonii×P. nigra, Populus deltoides×euramericana 'Nanlin895', Populus alba×Populus glandulosa '84K', Populus alba var. pyramidalis Bunge, Populus davidiana×alba var. pyramidalis, and Populus alba×P. berolinensis, can all be obtained from Northeast Forestry University.

[0035] The plant materials were cultured in a phytotron, and the culture conditions were set as follows: temperature 22±1°C, relative humidity 35±5%, light intensity 10,000 Lux, and photoperiod 16 h light / 8 h dark.

[0036] Under sterile conditions, the apical young buds (about 3 cm in length) of tissue-cultured seedlings were excised using a sterilized scalpel and inoculated into the rooting medium, and subcultured every 14 days. The tissue-cultured seedlings with good growth status were selected as experimental materials for subsequent hairy root induction experiments.

[0037] The vectors used in the present invention are as follows:

[0038] 1step Cas9 ATU6 EGFP (self-modified, see the map in Figure 2 ) and 1step Cas9 ATU6 mCherry (self-modified, see the map in Figure 3 ).

[0039] The strains used in the present invention are as follows:

[0040] Agrobacterium rhizogenes K599

[0041] The drugs used in the present invention are as follows:

[0042] Kanamycin (Kan), purchased from Sigma-Aldrich, product number 60615;

[0043] Streptomycin (Str), purchased from Sigma-Aldrich, product number T8902;

[0044] Cefotaxime (Cef), purchased from Sigma - Aldrich, catalog number C7039;

[0045] Trypone was purchased from BD Difco, catalog number 211705;

[0046] Yeast Extract was purchased from BD Difco, catalog number 212750;

[0047] Sodium chloride (NaCl) was purchased from Sigma - Aldrich, catalog number S9888;

[0048] Agar was purchased from Sigma - Aldrich, catalog number A1296;

[0049] WPM basal medium was purchased from PhytoTech, catalog number M519;

[0050] Sucrose was purchased from Sigma - Aldrich, catalog number S7903;

[0051] MES buffer was purchased from Sigma - Aldrich, catalog number M3671;

[0052] Acetosyringone (As) was purchased from Sigma - Aldrich, catalog number D134406;

[0053] Plant hormone: Thidiazuron (TDZ) was purchased from Sigma - Aldrich, catalog number P6186.

[0054] The culture media involved in the present invention are as follows:

[0055] LB medium: 10 g / L Trypone, 5 g / L Yeast Extract, 5 g / L NaCl, 15 g / L Agar, the balance being water.

[0056] Poplar co - culture medium: 2.41 g / L WPM, 25 g / L Sucrose, 0.5 g / L MES, 6 g / L Agar, the balance being water, pH = 5.8. Add 100 μM As after sterilization.

[0057] Poplar rooting medium: 2.41 g / L WPM, 25 g / L Sucrose, 0.5 g / L MES, 6 g / L Agar, the balance being water, pH = 5.8. Add 200 mg / L Cef after sterilization.

[0058] Poplar budding induction medium: 2.41 g / L WPM, 25 g / L sucrose, 0.5 g / L MES, 0.02 mg / L TDZ, 6 g / L agar, with the balance being water, pH = 5.8. Add 200 mg / L Cef after sterilization.

[0059] The present invention provides a method for establishing a non-genotype-dependent root-tiller regeneration transformation system of woody plants mediated by Agrobacterium rhizogenes. In the experiments of inducing transgenic hairy roots and plant regeneration, the research is divided into two key stages: the stage of hairy root induction and transgenic verification, and the stage of plant regeneration. In the first stage, we used the transformation method mediated by Agrobacterium rhizogenes to genetically transform the apical buds of poplar. Specifically, the poplar explants were immersed in the Agrobacterium rhizogenes suspension (OD 600nm = 0.3 - 0.4) containing the binary vector. After 2 days of co-culture, they were transferred to the hairy root induction medium without antibiotics. Under these conditions, the explants could produce more than 6 independent roots on average. To ensure the accurate screening of transgenic roots, we used the mCherry fluorescent reporter gene for visual screening, which effectively avoided the interference of non-transgenic roots. To further verify the transgenic integration, PCR technology was used to perform molecular detection on the roots. The results confirmed that the mCherry gene had been successfully integrated into the target genome and excluded the possibility of Agrobacterium tumefaciens contamination. In the second stage, the confirmed transgenic hairy roots were used for the plant regeneration experiment. First, all non-transgenic roots were removed. Subsequently, the transgenic hairy roots were cut into root segments of 1 - 2 cm and inoculated in the TDZ budding induction medium. After about 2 months of culture, the morphological transformation from hairy roots to buds was successfully achieved. The obtained transgenic buds were transferred to the rooting medium, and the formation of new roots could be observed about 1 week later, completing the entire regeneration process and obtaining transgenic plants.

[0060] Example 1: Method for establishing a non-genotype-dependent root-tiller regeneration transformation system of woody plants mediated by Agrobacterium rhizogenes

[0061] In this example, taking the PDS gene as the target gene, a non-genotype-dependent root-tiller regeneration gene editing system for woody plants is provided. PDS (phytoene desaturase gene) is a gene encoding pyridoxal synthase, which is a commonly used marker gene for testing the effectiveness of gene editing systems. The PDS gene plays a role in the biosynthesis of carotenoids. When a loss-of-function mutation occurs in this gene, it will cause the plant to be unable to synthesize normal carotenoids, usually manifested as obvious bleaching of the plant. This visual change enables researchers to quickly and intuitively judge whether gene editing is successful. The non-genotype-dependent root-tiller regeneration gene editing system for woody plants provided in this example is as follows:

[0062] 1. Construction of the vector

[0063] In this study, the pCAMBIA1300 vector was used as a backbone for modification. The resistance gene was removed by enzymatic digestion, and under the regulation of the 35S promoter, the fluorescent marker genes EGFP (enhanced green fluorescent protein) and mCherry (red fluorescent protein) were inserted respectively for the screening and observation of transgenic plants (the schematic diagram of vector construction is as shown in Figure 1 ). The modified vectors were named 1step Cas9 ATU6 EGFP (see Figure 2 ) and 1step Cas9 ATU6 mCherry (see Figure 3 ).

[0064] Based on the reported coding region sequence of the PDS gene in Populus tomentosa, an efficient 20bp target sequence was screened, and it was ensured that the PAM sequence (NGG) was located downstream of the sgRNA target site. The finally selected target site was: sgRNA: 5'-GTGTTATCAAGGTCCGGTCT-3' (the nucleotide sequence is as shown in SEQ ID NO.1) (PAM: TGG). A sticky end sequence identical to the BsaⅠ cleavage site was added in front of the sgRNA sequence as the sgRNA primer (see Table 1), and it was annealed from 98℃ to room temperature to form double-stranded DNA with sticky ends. The annealed sgRNA was placed in a 4℃ refrigerator for subsequent ligation use.

[0065] Table 1 Information table of sgRNA primers

[0066]

[0067] The 1step Cas9 ATU6 EGFP and 1step Cas9 ATU6 mCherry plasmids were digested with BsaⅠ. The samples were placed in a PCR instrument for enzymatic digestion at 37℃ for 2h and inactivated at 65℃ for 20min. The digested linearized vectors were ligated with the sgRNA diluted 200 times through T4 ligase, and ligated at room temperature for 10min. Subsequently, the recombinant vectors were transformed into Escherichia coli DH5α competent cells for amplification, and the correct construction of the vectors was verified by DNA sequencing to ensure the accuracy and integrity of the vector structure.

[0068] 2. Inducing transgenic poplar hairy roots by Agrobacterium rhizogenes

[0069] Under sterile conditions, pick a single colony of transformed Agrobacterium rhizogenes and inoculate it into 4 mL of LB liquid medium, adding 4 μL of Kan (50 mg / mL) and 4 μL of Str (50 mg / mL). Incubate overnight in a constant temperature shaking incubator at 28 °C and 220 r / min. Subsequently, transfer the bacterial solution to 50 mL of LB liquid medium (containing antibiotics: 2.5 mg Kan, 2.5 mg Str) and continue culturing until the OD 600nm value reaches 0.6 for standby.

[0070] Select Populus trichocarpa, Populus ussuriensis, Populus bolleana, Populus deltoides cv. 'Nanlin895', Populus alba×Populus glandulosa '84K', Populus simonii×Populus nigra, Populus davidiana×Populus bolleana, and Populus alba×Populus berolinensis plants aged 2 - 8 weeks. Under sterile conditions, cut the apical buds at the 4th - 5th stem nodes, remove the excess leaves, and immerse the cut ends into the Agrobacterium rhizogenes K599 bacterial solution for 15 minutes. Then, blot dry the residual bacterial solution on the surface with sterile filter paper.

[0071] Insert the infected stem segments into the co - culture medium and co - culture them at 25 ± 2 °C for 2 days (see Figure 4 ). After co - culture, wash them 3 - 5 times with sterile water containing cephalosporin (Cef), 30 minutes each time, and finally blot dry the moisture with sterile filter paper.

[0072] Transfer the co - cultured stem segments to the WPM basal medium containing 400 mg / L Cef and place them under the following culture conditions: light cycle 16 h / 8 h (light / dark), temperature 25 ± 2 °C, relative humidity 50 - 60%. After 8 days of culture, the base of the stem segments begins to swell; after 11 days of culture, visible white adventitious roots are formed; after 30 days of culture, a large number of white roots are formed (see Figure 4 ).

[0073] 3. Fluorescence signal detection of poplar hairy roots

[0074] To verify the expression of foreign genes in transgenic hairy roots, a stereoscopic fluorescence microscope (Zeiss) was used to detect the expression of EGFP and mCherry fluorescence - labeled genes. The specific steps are as follows:

[0075] After 30 days of hairy root induction culture, take out the roots from the medium and wash them with sterile water to remove the residual bacteria and medium on the surface. Place the washed samples in a sterile petri dish, place the petri dish on the stage, and conduct fluorescence microscope observation. Fluorescence detection parameter settings: mCherry: excitation wavelength 587 nm, emission wavelength 610 nm; EGFP: excitation wavelength 488 nm, emission wavelength 510 nm.

[0076] By observing the fluorescence signal, it is determined whether the exogenous vector has been transferred into the hairy roots. Roots that emit red fluorescence at an excitation wavelength of 587 nm or green fluorescence at an excitation wavelength of 488 nm are transgenic root systems (see Figure 5 ).

[0077] 4. Induction of adventitious buds from hairy roots

[0078] The transgenic hairy roots are aseptically transferred to the bud induction medium. Callus masses can grow out after about 14 days of culture. After continuing to culture on the bud induction medium for about 9 days, the callus masses begin to differentiate into bud tips. After culturing for about 21 more days, young buds of 2 - 3 cm in length are formed (see Figure 6 ). Subsequently, select healthy young buds (about 2 cm in length) and transfer them to the rooting medium. Continue to culture for 2 - 3 weeks. After the root system develops completely, complete transgenic plants are obtained.

[0079] Precautions:

[0080] 1. Ensure complete removal of Agrobacterium;

[0081] 2. The treatment process should be gentle to prevent damage to the roots.

[0082] To establish a widely applicable poplar genetic transformation system, the present invention selected 8 poplar germplasms with important economic value as experimental materials. Through systematic screening and optimization, we tested a variety of basic media (MS, WPM, B5) and their modified formulations, and evaluated the effects of different plant growth regulators (IAA: 0.1 - 1.0 mg / L; IBA: 0.05 - 0.5 mg / L; BAP: 0.1 - 2.0 mg / L; GA3: 0.01 - 0.1 mg / L; Zeatin: 0.1 - 1.0 mg / L) on bud regeneration. The experimental results show that normal WPM with only sucrose added can be used as the rooting medium to make poplar take root.

[0083] In the experiment, the present invention tried 97 different culture medium formulations with different ratios of auxin and cytokinin (the basic formulation of this medium is 2.41 g / L WPM, 25 g / L sucrose, 0.5 g / L MES, 6 g / L agar, the balance is water, pH = 5.8. After sterilization, add 200 mg / L Cef.) for the test of root - regenerated bud transformation. We counted the bud induction efficiency according to the proportion of buds emerging from different root segments. If all the tested root segments can induce buds, it means a 100% bud induction efficiency. We input all the culture medium formulations and their corresponding root - induced bud efficiencies into the matlab software for modeling analysis. We can find that when the exogenous auxin is 0 and the cytokinin TDZ concentration is (0.02 mg / L) in the WPM basic medium, the maximum bud induction rate (100%) can be obtained (see Figure 7)。The effects of the formulations of other bud induction media are not as good as that of this formulation. Through this model, we found that with the increase in the concentration of externally applied auxin, the efficiency of bud induction will decrease. Therefore, the final formulation of the bud induction medium selected in the present invention is 2.41 g / L WPM, 25 g / L sucrose, 0.5 g / L MES, 0.02 mg / L TDZ, 6 g / L agar, with the balance being water, pH = 5.8, and adding 200 mg / L Cef after sterilization. This bud induction medium formulation can efficiently induce bud regeneration from the roots of various poplar species, with an average regeneration rate of 100%( Figure 7 )。Further optimization shows that this medium formulation exhibits good applicability in 8 poplar germplasms. The regenerated buds show normal morphological characteristics and growth potential, providing high-quality materials for subsequent genetic transformation experiments.

[0084] To evaluate the editing efficiency of the CRISPR / Cas9 system in poplar, the present invention used the PDS (Phytoene desaturase) gene as a target for functional verification. The experimental data show that in 8 poplar germplasms, the average editing efficiency of the PDS gene reaches 39.2% (see Figure 8 )。Further analysis of the editing types reveals that CRISPR / Cas9 mainly induces three mutation patterns (see Figure 9 ):deletion of 2 bases, deletion of 1 base, and insertion of 1 base. These mutations all lead to a frameshift in the reading frame of the PDS gene, generating a premature termination codon (PTC), thereby blocking the biosynthesis pathway of carotenoids. Phenotypic analysis shows that the transgenic plants with successful editing of the PDS gene all exhibit a typical albino phenotype ( Figure 8 ), which is consistent with the expected results. It is worth noting that there are significant differences in the editing efficiency among different poplar germplasms (P < 0.05), among which the editing efficiency of Populus simonii × P. nigra is the highest (64.5%), while that of Populus ussuriensis is relatively low (18.9%). This difference may be related to the genomic background and DNA repair mechanisms of different germplasms and is worthy of further study.

[0085] It should be noted that the above embodiments are only used to illustrate the technical solutions 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 preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. A method for establishing an Agrobacterium rhizogenes-mediated genotype-independent root sucker regeneration transformation system for woody plants, characterized in that, The method includes the following steps: S1. Transfer the expression vector containing the target gene into Agrobacterium rhizogenes to obtain Agrobacterium rhizogenes containing the target gene; the expression vector containing the target gene is obtained by using the pCAMBIA1300 vector as the backbone, removing the resistance gene, and inserting the EGFP or mCherry fluorescent marker gene under the regulation of the 35S promoter. S2. Cut the apical bud of the poplar tissue culture seedling, immerse the incision part in the Agrobacterium rhizogenes bacterial solution for infection, insert it into the co-culture medium after infection for co-culture. After the co-culture is completed, wash it 3 - 5 times with sterile water containing Cef, transfer the washed stem segment to the WPM basal medium containing Cef, and after culturing for 30 days, a large number of white roots are formed, which are hairy roots. S3. Use a stereoscopic fluorescence microscope to detect the expression of the EGFP or mCherry fluorescent marker gene in the hairy roots obtained in S2. S4. Transfer the transgenic hairy roots aseptically to the shoot induction medium, culture for 34 - 56 days to obtain young shoots, select the vigorously growing young shoots and transfer them to the rooting medium, and continue to culture for 2 - 3 weeks. After the roots are fully developed, obtain complete transgenic plants; the composition of the shoot induction medium is 2.41 g / L WPM, 25 g / L sucrose, 0.5 g / L MES, 0.02 mg / L TDZ, 6 g / L agar, the balance is water, pH = 5.8, and add 200 mg / L Cef after sterilization.

2. The establishment method according to claim 1, characterized in that The Agrobacterium rhizogenes described in S1 is K599.

3. The establishment method according to claim 1, characterized in that The poplar varieties described in S2 include Populus trichocarpa, Populus ussuriensis, Populus simonii×Populus nigra, Nanlin 895 Poplar, 84K Poplar, Populus alba var. pyramidalis, Populus davidiana×Populus bolleana, and Populus alba×Populus berolinensis.

4. The establishment method according to claim 1, wherein The OD of the Agrobacterium rhizogenes bacterial solution described in S2 600nm is 0.6, and the infection time is 15 min.

5. The establishment method according to claim 1, characterized in that The composition of the co-culture medium described in S2 is 2.41 g / L WPM, 25 g / L sucrose, 0.5 g / L MES, 6 g / L agar, the balance is water, pH = 5.8, and add 100 μM As after sterilization.

6. The establishment method according to claim 1, characterized in that The co-culture conditions described in S2 are 25 ± 2 °C for 2 days.

7. The establishment method according to claim 1, characterized in that, The concentration of Cef in the WPM basal medium described in S2 is 400 mg / L, and the corresponding culture conditions of the WPM basal medium are 16 h of light, 8 h of darkness, temperature 25 ± 2 °C, and relative humidity 50 - 60%.

8. The establishment method according to claim 1, wherein The specific steps of S3 are as follows: Wash the hairy roots with sterile water and observe them under a fluorescence microscope. The fluorescence detection parameters are set as follows: the excitation wavelength of mCherry is 587 nm, and the emission wavelength is 610 nm; the excitation wavelength of EGFP is 488 nm, and the emission wavelength is 510 nm. By observing the fluorescence signal, judge whether the foreign vector has been transferred into the hairy roots. The roots that emit red fluorescence at the excitation wavelength of 587 nm or green fluorescence at the excitation wavelength of 488 nm are transgenic roots.

9. The establishment method according to claim 1, characterized in that, The composition of the rooting medium described in S4 is 2.41 g / L WPM, 25 g / L sucrose, 0.5 g / L MES, 6 g / L agar, the balance is water, pH = 5.8, and add 200 mg / L Cef after sterilization.

10. Application of the establishment method according to any one of claims 1 - 9 in the breeding of woody plants.

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