A non-tissue culture dependent agrobacterium rhizogenes mediated genetic transformation method of pinus massoniana hairy roots

Abstract

The application discloses a non-tissue culture dependent Agrobacterium rhizogenes mediated genetic transformation method of Pinus massoniana hairy roots and belongs to the technical field of plant genetic transformation. A plasmid carrying RUBY and EGFP fluorescent reporter genes is transferred into Agrobacterium rhizogenes and an infection solution is prepared; after a Pinus massoniana seedling is cut, the cut is immersed into the infection solution and vacuum infiltration treatment is carried out, the root wound is wrapped with the bacteria and then the seedling is transplanted and hairy roots are induced; and through observation of the color and EGFP fluorescent signal of the regenerated hairy roots, a transgenic Pinus massoniana plant is efficiently screened. The application constructs a high-efficiency and stable genetic transformation system of Pinus massoniana, the system can induce a composite plant containing transgenic roots without experiencing a complicated tissue culture process under non-aseptic conditions, effectively solves the technical problem of the lack of a genetic transformation system of Pinus massoniana roots, and provides technical support for Pinus massoniana gene function research, secondary metabolite development and genetic improvement.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic transformation technology, specifically relating to a non-tissue culture-dependent Agrobacterium rhizogenes-mediated genetic transformation method for the hairy roots of Pinus massoniana. Background Technology

[0002] Masson pine (Pinus massoniana) is an important pioneer tree species for community succession in the subtropical low mountain and hilly areas of my country. It possesses excellent characteristics such as rapid growth, high yield, strong adaptability, and tolerance to poor soil, playing a vital role in the stability of the southern mountain ecosystem, soil and water conservation, and the cultivation of timber forests. With the continuous advancement of the high-quality development strategy of forestry and the construction of ecological civilization, production practices have placed higher demands on traits such as growth rate, timber quality, disease and pest resistance, and stress adaptability of Masson pine. However, current germplasm innovation in Masson pine still mainly relies on traditional selection and hybridization breeding methods, which suffer from problems such as long breeding cycles, slow genetic gain, difficulty in accurately aggregating superior alleles, and low efficiency in improving target traits, failing to meet the urgent need of modern forestry for rapid and targeted improvement of superior varieties. Therefore, utilizing genetic engineering technology to achieve targeted improvement of important traits has become an important development direction for modern bio-breeding of Masson pine.

[0003] Plant genetic transformation technology is an important means of introducing exogenous genes, validating their function, and improving molecular structure. Currently, commonly used plant genetic transformation methods mainly include Agrobacterium-mediated transformation and gene gun methods. Traditional plant genetic transformation methods typically rely on strict aseptic conditions and cumbersome tissue culture and regeneration systems, resulting in problems such as complex procedures, long culture cycles, high costs, and strong dependence on explant type and genotype. This is especially true for coniferous species, where the establishment of genetic transformation systems is even more challenging due to difficulties in constructing regeneration systems, low callus induction efficiency, and susceptibility to browning.

[0004] Agrobacterium rhizogenes-mediated hairy root induction technology can integrate T-DNA into the plant genome via its Ri plasmid, directly inducing genetically stable hairy roots without the need for complete plant regeneration. It features relatively simple operation, short induction cycle, and high transformation efficiency, and has been applied in various fields such as functional gene analysis, metabolic regulation research, elucidation of secondary metabolite synthesis mechanisms, and verification of gene editing effects in plants.

[0005] However, an efficient and stable genetic transformation system for Masson pine has not yet been established, and related research is still in the exploratory stage. Existing studies largely rely on model plants such as Arabidopsis thaliana and tobacco for heterologous expression verification, which makes it difficult to accurately reflect the expression regulation characteristics and biological functions of target genes in Pinus species, thus limiting the in-depth development of related functional gene research and molecular breeding work. Therefore, establishing a method that can rapidly induce hairy roots in Masson pine and achieve stable genetic transformation without relying on complex tissue culture systems is of great significance for constructing an efficient molecular function verification platform for Masson pine, accelerating the innovation of superior germplasm, and promoting the establishment of a modern bio-breeding technology system. Summary of the Invention

[0006] To address the aforementioned shortcomings in existing technologies, this invention provides a non-tissue culture-dependent Agrobacterium rhizogenes-mediated genetic transformation method for hairy roots of Pinus massoniana. This method overcomes the technical bottleneck of difficult genetic transformation of Pinus massoniana by optimizing the infection system and culture conditions, and establishes a simple, short-cycle, and callus-regeneration-free hairy root genetic transformation method, providing a stable and efficient technical platform for functional gene research and molecular breeding of Pinus massoniana.

[0007] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows: A tissue culture-independent Agrobacterium rhizogenes-mediated genetic transformation method for the hairy roots of Pinus massoniana, comprising the following steps: (1) Transform the recombinant expression vector containing the visual reporter gene into Agrobacterium rhizogenes to obtain an engineered strain of Agrobacterium rhizogenes carrying the visual reporter gene; (2) After activation and expansion culture, the engineered strain of Agrobacterium rhizogenes was centrifuged to collect the bacterial cells. The bacterial cells were resuspended twice with MESKOH resuspension containing 2-morpholine ethanesulfonic acid and magnesium chloride. Acetyl eugenol was added and the mixture was allowed to stand at room temperature in the dark to obtain the infection solution. (3) Select seedlings of Masson pine, cut the roots at the junction of the root and stem, immerse the wound in the inoculum solution for infiltration treatment; remove the seedlings and remove excess bacterial solution from the surface, wrap the engineered bacteria at the wound, transplant them into a moist substrate, and induce the production of hairy roots. (4) After rooting, the positive hairy roots are initially screened by the appearance traits of the visual reporter gene expression. Then, the visual reporter gene is verified by PCR amplification. Plants containing the visual reporter gene are successfully transformed plants.

[0008] Furthermore, in step (1), the recombinant expression vector contains a RUBY visual reporter gene and an EGFP fluorescent reporter gene expressed driven by the CaMV 35S promoter.

[0009] Furthermore, the Agrobacterium rhizogenes mentioned in step (1) is strain K599.

[0010] Further, the activation and expansion culture process in step (2) is as follows: the activated Agrobacterium rhizogenes engineered strain is inoculated into 1.5 mL of TY liquid medium containing 45-55 mg / L kanamycin and cultured with shaking for 11-13 h. Then, 100-500 μL of the bacterial solution is transferred to 30 mL of TY liquid medium containing the same concentration of kanamycin and cultured until the bacterial solution reaches OD. 600 A value of 0.9 to 1.4 is sufficient.

[0011] Furthermore, the TY liquid medium used in step (2) for activation and expansion culture contains 5 g peptone, 3 g yeast extract and 1 M calcium chloride per liter; the TY solid medium is supplemented with 15 g agar powder.

[0012] Furthermore, the MESKOH resuspension in step (2) contains 10 mM 2-morpholinoethanesulfonic acid and 10 mM magnesium chloride.

[0013] Furthermore, in step (2), the concentration of acetylsylgenone in the inoculum is 180~200 μmol / L, and the standing time is 0.5~1.5h.

[0014] Further, in step (3), select two-week-old seedlings of Pinus massoniana whose seed coats have completely fallen off, and cut the roots within 0.5 cm above and below the root-stem junction.

[0015] Furthermore, in step (3), the permeation treatment is carried out under a vacuum pressure of 0.04~0.06 MPa for 4~16 min.

[0016] Furthermore, in step (3), the engineered bacterial cells wrapped around the wound are bacterial precipitates collected by centrifugation after two activations and expansion cultures of a single positive colony.

[0017] Furthermore, the matrix used in step (3) is vermiculite with a particle size of 2-5 mm.

[0018] Furthermore, during root induction in step (3), the plants are first cultured in the dark under high humidity, and then cultured under light conditions. The dark culture conditions are: temperature 23~27℃, relative humidity 60~80%, culture time 1~2 days. The light culture conditions are: temperature 23~27℃, photoperiod 16 hours light / 8 hours dark, humidity 60~80%, light intensity 150~200 μmol·m -2 ·s -1 .

[0019] The beneficial effects of this invention are as follows: 1. This invention establishes for the first time a tissue culture-independent genetic transformation system for the hairy roots of Pinus massoniana, which eliminates the need to construct callus tissue and plant regeneration systems, avoiding the browning, contamination, and regeneration difficulties that are prone to occur during long-term tissue culture, and significantly reducing the technical threshold and experimental costs.

[0020] 2. The present invention optimizes key infection parameters, including strain type, bacterial concentration, vacuum permeation treatment time, and acetylsuccinone concentration, so as to achieve the best balance between bacterial activity and infection ability, thereby improving transformation efficiency while effectively reducing tissue necrosis and improving the stability and repeatability of the system.

[0021] 3. This invention combines the RUBY visual reporter gene with the EGFP fluorescent reporter system for dual screening, realizing a positive identification method that combines visually visible red betaine pigment primary screening with fluorescence detection secondary screening. It can perform preliminary screening without relying on expensive large-scale imaging equipment, greatly improving screening efficiency and reducing detection costs. At the same time, it combines PCR molecular verification to ensure the accuracy and reliability of genetic transformation results.

[0022] 4. The hairy root transformation system of Masson pine established in this invention can be widely used in research such as functional gene verification, promoter activity analysis, metabolic pathway research, transcriptional regulation mechanism analysis and superior germplasm creation, providing important technical support for the innovation of superior Masson pine germplasm and the establishment of a modern biological breeding technology system. Attached Figure Description

[0023] Figure 1 The plasmid map used in the embodiments of the present invention; Figure 2 This is a schematic diagram of the operation process for inducing hairy roots of Pinus massoniana in an embodiment of the present invention; Figure 3 This is a phenotypic diagram of the positive hairy root of the present invention; Figure 4 This is a fluorescence detection image of EGFP in the positive hairy roots of this invention; Figure 5 This is an electrophoresis image showing the relative expression level analysis and PCR detection of the RUBY reporter gene in positive hairy roots of this invention; wherein, Figure 5 A represents the results of relative expression analysis, CK represents the root system of the control group, and Y1~Y4 represent the positive hairy roots; Figure 5 B is the electrophoresis image of PCR detection, M is the DNA molecular weight marker, Plasmid is the positive control (RUBY plasmid), WT is the negative control (wild-type root system), and Y1~Y7 are positive hairy root samples of Masson pine. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described embodiments are merely some embodiments of the invention, and not all embodiments.

[0025] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0026] It should be noted that relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0027] The features and performance of the present invention will be further described in detail below with reference to the embodiments and accompanying drawings.

[0028] Example 1

[0029] (1) Preparation of Agrobacterium rhizogenes infection solution Thaw competent Agrobacterium rhizogenes cells K599 stored at -80℃ in an ice-water bath. Under aseptic conditions, add 300 ng of a plant expression vector carrying the RUBY and EGFP reporter genes to the competent cells. Gently pipette and mix well. Incubate in an ice-water bath for 5 min, flash freeze in liquid nitrogen for 5 min, incubate in an ice-water bath at 37℃ for 5 min, and incubate in an ice-water bath for 5 min. Then, add 700 μL of antibiotic-free TY liquid medium at room temperature and incubate at 28℃ with shaking at 200 rpm for 2-3 h. After centrifugation, discard part of the supernatant and keep about 100 μL of supernatant. Gently pipette and resuspend the bacterial block and spread it on TY solid medium containing 50 mg / L kanamycin. Invert and incubate at 28℃ for 2-3 days.

[0030] Select monoclonal colonies that tested positive by PCR and inoculate them into 1.5 mL of TY liquid medium containing 50 mg / L kanamycin. Incubate at 28 °C and 200 rpm with shaking for 12 h. Add 500 μl of the above bacterial culture to 30 mL of TY liquid medium containing the same concentration of kanamycin, and expand the culture to the OD value. 600 The OD value was adjusted to 0.8–1.5. The bacterial culture was centrifuged at 7000 rpm for 10 min at room temperature, the supernatant was discarded, and 30 mL of MES-KOH resuspension containing 10 mM 2-morpholine ethanesulfonic acid and 10 mM magnesium chloride was added to the collected bacterial cells. The cells were washed repeatedly by pipetting. The culture was centrifuged again under the same conditions, the supernatant was discarded, and the bacterial cells were resuspended in an appropriate amount of MES-KOH resuspension. The OD value was adjusted. 600 Add acetylsuccinone to a final concentration of 200 μM, and let stand at room temperature in the dark for 1 h to obtain Agrobacterium rhizogenes infection solution.

[0031] (2) Cultivation of Masson pine seedlings Select plump and healthy Masson pine seeds, soak them in a 0.5% potassium permanganate solution for 30 minutes to disinfect them, rinse them thoroughly with clean water, and then soak them in warm water with an initial temperature of 42℃. Change the water every 6 to 8 hours to prevent oxygen deficiency and microbial growth.

[0032] Once the seeds have sprouted extensively, sow them in a layer of pre-watered, moist but not waterlogged vermiculite (2-5 mm in diameter) and place them in a greenhouse for cultivation. The cultivation conditions are: temperature 25±2℃, photoperiod of 16 h light / 8 h darkness, and relative humidity of 40%~60%.

[0033] Once the seedlings have grown to 2 weeks old and the seed coat has completely fallen off, select healthy Masson pine seedlings. Use a sterile blade to cut the roots at the junction of the rootstock, making an incision at approximately a 45° angle to increase the contact area with the infection solution.

[0034] (3) Agrobacterium rhizogenes infection and hairy root induction After root cutting, the wound ends of the seedlings were immersed in the infection solution prepared in step (1) and treated under a vacuum of 0.06 MPa for 12 min. After removal, residual bacterial solution was absorbed using sterile filter paper, and centrifuged Agrobacterium rhizogenes precipitate was wrapped around the wound. The seedlings were then transplanted into seedling pots containing moist vermiculite and cultured in the dark for 2 days to promote stable infection. Subsequently, they were transferred to normal light conditions for continued culture to induce hairy root formation. During the culture period, a 1:800 diluted Hoagland solution was sprayed every 5 days to supplement nutrients, and the substrate was kept moist but not waterlogged. The culture conditions were: temperature 25±2℃, photoperiod 16 h light / 8 h dark, humidity 60-80%.

[0035] Typically, after being transferred to a greenhouse for cultivation, hairy root primordia begin to form at the wound site after about 20 days, and clearly visible hairy roots begin to form after 25 to 50 days.

[0036] (4) Identification of transgenic hairy roots After 30-50 days of induction, seedlings were carefully removed and the roots were rinsed with sterile water. Hairy roots exhibiting a distinct red betaine pigment deposition phenotype were visually selected as primary positive materials. To eliminate interference from root background color and gene expression levels, the primary screening materials were placed in a dark room and a handheld EGFP excitation light source was used to detect green fluorescence signals for secondary screening. Materials exhibiting both a red phenotype and green fluorescence signals were selected as candidate transformed root systems. The plasmid maps used are shown below. Figure 1 The induction process is shown in Figure 2 Positive hairy root phenotype is seen Figure 3 Positive hairy roots with EGFP are observed. Figure 4 .

[0037] Roots of control (WT) plants and candidate transformed plants were excised using a sterile blade, and genomic DNA and total RNA were extracted using the CTAB method. The extracted DNA was identified by PCR using RUBY gene-specific primers RUBY-F and RUBY-R. RUBY-F: 5'-CTTGAGTCCTCCGAGAAGCC-3' (SEQ ID NO.1); RUBY-R: 5'-TCACTGGAGGCTTGGCTCA-3' (SEQ ID NO.2); the PCR reaction system used is shown in Table 1; the PCR reaction procedure used is shown in Table 2.

[0038] Table 1 PCR reaction system

[0039] Table 2 PCR reaction procedure

[0040] The PCR products were detected by 1% agarose gel electrophoresis, and the results are shown in the attached figure. Figure 5 As shown in B, both the root system of the candidate transformed plants and the plasmid control amplified a RUBY gene-specific band of approximately 564 bp, consistent with the expected size, while the untransformed control (WT) root system did not show this band.

[0041] The total RNA extracted was reverse transcribed to obtain cDNA, and the expression level of the RUBY reporter gene was detected by real-time quantitative PCR (qPCR). qPCR was performed using BioRad's iTaq™ Universal SYBR® Green Supermix fluorescent dye on a CFX96™ real-time quantitative PCR instrument. The UBC gene from *Pinus massoniana* was used as an internal control, and three technical replicates were set for each sample. Data were analyzed using a 23... -ΔCt The relative expression levels were calculated using the following methods. The primer sequences for qPCR detection are shown in Table 3.

[0042] Table 3 Primer sequences for qPCR detection

[0043] The results are attached. Figure 5 As shown in Figure A, compared with the untransformed control root system, the expression of the RUBY gene could be detected in the transgenic hairy roots, indicating that the RUBY gene had been successfully integrated and expressed.

[0044] Example 2

[0045] To further optimize the transformation system of this invention, this embodiment employs a single-factor experimental design to systematically compare key technical parameters to determine the optimal transformation conditions. The experimental material consisted of two-week-old Pinus massoniana seedlings after root cutting treatment. Except for the factors investigated, all other steps and culture conditions were performed as described in Example 1. The hairy root induction rate and positive transformation rate were calculated using the following formula: Induction rate (%) = (Number of hairy-rooted plants / Total number of infected plants) × 100%; Positive conversion rate (%) = (Number of positive plants / Total number of infected plants) × 100%.

[0046] (1) Effect of different strain types on transformation efficiency Agrobacterium rhizogenes strains K599, MSU440, C58C1, Ar.Qual, and Ar.1193 were selected, and the implementation method was the same as that described in Example 1.

[0047] The results are shown in Table 3. Significant differences in transformation efficiency were observed among different strains. The K599 strain treatment group exhibited the highest hairy root induction rate (46.88%) and a positive transformation rate of 12.63%, significantly higher than other strains. This indicates that strain type is one of the key factors affecting the induction of hairy roots and the efficiency of genetic transformation in *Pinus massoniana*.

[0048] Table 3. Effects of different bacterial strains on the induction rate of hairy roots of Pinus massoniana.

[0049] (2) Effect of bacterial concentration on transformation efficiency bacterial culture concentration OD 600 The values ​​were set to 0.6, 0.8, 1.0, 1.2, and 1.4 respectively, and the implementation method was the same as that described in Example 1.

[0050] The results are shown in Table 4. The bacterial concentration significantly affected the transformation efficiency. When the bacterial concentration OD... 600 When the value is 1.0, the positive conversion rate of hairy roots reaches its highest level, at 15.63%; OD 600 When the OD value is below 0.8, the bacterial count is insufficient, and the infection efficiency decreases; 600 At concentrations above 1.2, some plants showed significant necrosis, affecting subsequent hairy root formation. This indicates that an appropriate bacterial concentration helps balance infection efficiency and plant survival rate.

[0051] Table 4. Effects of different bacterial concentrations on the induction rate of hairy roots of Pinus massoniana.

[0052] (3) Effect of infection time on conversion efficiency The infection time was set to 4 min, 8 min, 12 min and 16 min respectively, and the implementation method was the same as that described in Example 1.

[0053] The results are shown in Table 5. The hairy root induction rate and positive conversion rate were highest when the infection time was 12 min. When the infection time was 4 min or 8 min, the infection contact time was insufficient and the positive conversion rate decreased significantly. When the infection time was extended to 16 min, some seedlings showed tissue damage, which led to a decrease in the effective conversion rate.

[0054] Table 5. Effects of different infection times on the induction rate of hairy roots of Pinus massoniana.

[0055] (4) Effect of acetylsyringone concentration on conversion efficiency The concentrations of acetylsylphenone were set at 100 μM, 150 μM, 200 μM, and 250 μM, respectively, and the infection treatment was carried out according to the steps in Example 1.

[0056] The results are shown in Table 6. The hairy root induction rate and positive transformation rate were highest in the 200 μM acetylsylenone treatment group. The transformation efficiency was relatively low in the 100 μM and 150 μM treatment groups, indicating that too low a concentration of acetylsylenone is not conducive to the activation of vir gene expression and T-DNA transfer.

[0057] Table 6. Effects of different acetylsyringone concentrations on the induction rate of hairy roots of Pinus massoniana.

[0058] In summary, this invention has established a highly efficient and stable Agrobacterium rhizogenes-mediated genetic transformation system for the hairy roots of Pinus massoniana by systematically optimizing key factors such as strain type, infection time, bacterial concentration, and acetylsyringone concentration.

[0059] It should be noted that the present invention has been comprehensively and systematically described through general description, specific embodiments, and experimental verification. The above embodiments are merely preferred embodiments and are not intended to limit the present invention. All equivalent substitutions or conventional technical improvements made within the scope of the technical concept of the present invention should be included within the protection scope of the present invention.

Claims

1. A non-tissue culture-dependent Agrobacterium rhizogenes-mediated genetic transformation method for the hairy roots of Pinus massoniana, characterized in that, Includes the following steps: (1) Transform the recombinant expression vector containing the visual reporter gene into Agrobacterium rhizogenes to obtain an engineered strain of Agrobacterium rhizogenes carrying the visual reporter gene; (2) After activation and expansion culture, the engineered strain of Agrobacterium rhizogenes was centrifuged to collect the bacterial cells. The bacterial cells were resuspended twice with MESKOH resuspension containing 2-morpholine ethanesulfonic acid and magnesium chloride. Acetyl eugenol was added and the mixture was allowed to stand at room temperature in the dark to obtain the infection solution. (3) Select seedlings of Masson pine, cut the roots at the junction of the root and stem, immerse the wound in the inoculum solution for infiltration treatment; remove the seedlings and remove excess bacterial solution from the surface, wrap the engineered bacteria at the wound, and transplant them into a moist vermiculite substrate to induce the production of hairy roots. (4) After rooting, the positive hairy roots are initially screened by the appearance traits of the visual reporter gene expression. Then, the visual reporter gene is verified by PCR amplification. Plants containing the visual reporter gene are successfully transformed plants.

2. The method for non-tissue culture-dependent Agrobacterium rhizogenes-mediated genetic transformation of Pinus massoniana hairy roots as described in claim 1, characterized in that, In step (1), the recombinant expression vector contains a RUBY visual reporter gene and an EGFP fluorescent reporter gene expressed driven by the CaMV 35S promoter.

3. The method for genetic transformation of Pinus massoniana hairy roots mediated by Agrobacterium rhizogenes without tissue culture dependence as described in claim 1, characterized in that, The Agrobacterium rhizogenes mentioned in step (1) is strain K599.

4. The method for genetic transformation of Pinus massoniana hairy roots mediated by Agrobacterium rhizogenes without tissue culture dependence as described in claim 1, characterized in that, The activation and expansion culture process in step (2) is as follows: The activated Agrobacterium rhizogenes engineered strain is inoculated into 1.5 mL of TY liquid medium containing 45-55 mg / L kanamycin and cultured with shaking for 11-13 h. Then, 100-500 μL of the bacterial solution is transferred to 30 mL of TY liquid medium containing the same concentration of kanamycin and the culture is expanded until the bacterial solution reaches OD. 600 A value of 0.9 to 1.4 is sufficient.

5. The method for non-tissue culture-dependent Agrobacterium rhizogenes-mediated genetic transformation of Pinus massoniana hairy roots as described in claim 1, characterized in that, Step (2) The MESKOH resuspension contains 10 mM 2-morpholinoethanesulfonic acid and 10 mM magnesium chloride.

6. The method for non-tissue culture-dependent Agrobacterium rhizogenes-mediated genetic transformation of Pinus massoniana hairy roots as described in claim 1, characterized in that, In step (2), the concentration of acetylsuccinone in the infiltration solution is 180~200 μmol / L, and the standing time is 0.5~1.5h.

7. The method for genetic transformation of Pinus massoniana hairy roots mediated by Agrobacterium rhizogenes without tissue culture dependence as described in claim 1, characterized in that, In step (3), select 2-week-old seedlings of Masson pine with completely shed seed coats, and cut the roots within 0.5cm above and below the root-stem junction.

8. The method for non-tissue culture-dependent Agrobacterium rhizogenes-mediated genetic transformation of Pinus massoniana hairy roots as described in claim 1, characterized in that, In step (3), the permeation treatment is carried out under a vacuum pressure of 0.04~0.06 MPa for 4~16 min.

9. The method for non-tissue culture-dependent Agrobacterium rhizogenes-mediated genetic transformation of Pinus massoniana hairy roots as described in claim 1, characterized in that, In step (3), the engineered bacteria wrapped around the wound is a bacterial pellet collected by centrifugation after two activations and expansion cultures of Agrobacterium rhizogenes.

10. The method for genetic transformation of Pinus massoniana hairy roots mediated by Agrobacterium rhizogenes without tissue culture dependence as described in claim 1, characterized in that, In step (3), during root induction, the plants are first cultured in the dark under high humidity, and then cultured under light conditions. The dark culture conditions are: temperature 23~27℃, relative humidity 60~80%, culture time 1~2 days. The light culture conditions are: temperature 23~27℃, photoperiod 16 hours light / 8 hours dark, humidity 60~80%, light intensity 150~200 μmol·m -2 ·s -1 .

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