Method for inducing transgenic root of cinnamomum camphora and application thereof

Abstract

The application discloses a kind of transgenic rootlet induction method of Drynaria fortunei and application thereof, the induction method includes the following steps: S1: picking the third to fourth piece of leaf under Drynaria fortunei terminal bud as explant;S2: preparation of Agrobacterium rhizogenes infection solution and Agrobacterium rhizogenes bacteria;S3: the leaf blade collected in step S1 is placed in the Agrobacterium rhizogenes infection solution obtained in step S2, cut off petiole, expose leaf base, the Agrobacterium rhizogenes bacteria obtained in step S2 is applied in leaf base, to obtain the explant leaf base after inoculation;S4: the explant leaf base after inoculation in step S3 is inserted into sterilized culture medium and is cultured, i.e. hairy root can be induced;The culture medium includes vermiculite and soil.The induction method culture condition is simple, easy to operate, and the transgenic rootlet induction rate is greater than 80% by PCR detection, the induction method effectively breaks through the technical barrier of Drynaria fortunei transgenic system establishment, and the cost is low using pure vermiculite culture medium.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a method for inducing transgenic hairy roots of camphor tree and its application. Background Technology

[0002] Borneol, also known as "dragon brain" or "plum blossom," is a traditional Chinese medicine and precious natural spice with a long history and wide range of uses. Due to its natural scarcity and remarkable efficacy, it is in high demand in the pharmaceutical, high-end skincare, and fragrance industries. Currently, most borneol on the market is chemically synthesized (e.g., from turpentine oil), usually a mixture of L- and D-borneol (racemic mixture), which is considered far inferior to natural borneol in terms of purity, safety, and efficacy. Borneol camphor (… Cinnamomum camphora *Cinnamomum camphora* var. *borneol*, a rare variety of the genus *Cinnamomum* in the Lauraceae family, is the most important plant resource for extracting natural borneol. However, *Cinnamomum camphora* has strict requirements for light, temperature, and humidity, and is intolerant of drought and poor soil, limiting its suitable planting areas. Furthermore, under current cultivation techniques, the borneol yield of this plant is generally low. To overcome this industry bottleneck, cultivating high-quality *Cinnamomum camphora* plant resources has become an urgent task.

[0003] In recent years, molecular breeding techniques, especially transgenic and gene-editing technologies, have been successfully applied to many important economic crops, providing a highly promising technical path for the germplasm improvement of *Cinnamomum camphora*. However, a fundamental technical bottleneck hinders the application of molecular breeding methods in *Cinnamomum camphora*: to date, this species still lacks a stable and efficient genetic transformation system. Without reliable and mature technical methods to introduce exogenous genes into *Cinnamomum camphora* and obtain stably heritable transgenic plants, precision breeding techniques such as transgenic and gene-editing cannot be implemented.

[0004] Genetic transformation in most plants is Agrobacterium-dependent. Among them, Agrobacterium rhizogenes (… Agrobacterium rhizogenes As a Gram-negative bacterium belonging to the Rhizobium family, Agrobacterium rhizogenes exhibits excellent host versatility, capable of infecting various medicinal and economic crops, including cucumbers, melons, and salvia miltiorrhiza. It can also induce transgenic hairy root formation under conditions without exogenous hormones, significantly simplifying traditional transformation processes. Currently, Agrobacterium rhizogenes infection strategies mainly fall into two categories: ① aseptic explant inoculation; ② live plant injection / smearing. The latter, due to the lack of an aseptic operating environment, has advantages in cost control and technology dissemination. It is worth noting that the efficiency of hairy root induction is closely related to the selection of explants. Currently, conventional materials such as root-cut seedlings and hypocotyls are mainly used, while special explants such as girdled trunks are often used for woody plants. Summary of the Invention

[0005] This invention aims to solve at least one of the technical problems existing in the prior art. To this end, this invention proposes a method for inducing transgenic hairy roots of camphor tree. This induction method has simple culture conditions, is easy to operate, and PCR detection shows that the induction rate of transgenic hairy roots is >80%. This induction method effectively breaks through the technical barriers to establishing a transgenic camphor tree system. The culture method is simple, using pure vermiculite culture medium and water irrigation, and the cost is low.

[0006] The present invention also proposes the application of the above-mentioned induction method.

[0007] According to a first aspect of the present invention, a method for inducing transgenic hairy roots of camphor tree is provided, the induction method comprising the following steps:

[0008] S1: Collect the third to fourth leaves below the terminal bud of camphor tree as explants;

[0009] S2: Preparation of Agrobacterium rhizogenes infection solution and Agrobacterium rhizogenes cells;

[0010] S3: Place the leaves collected in step S1 into the Agrobacterium rhizogenes infection solution obtained in step S2, cut off the petiole to expose the leaf base, and apply the Agrobacterium rhizogenes bacteria obtained in step S2 to the leaf base to obtain the inoculated explant leaf base.

[0011] S4: Insert the leaf base of the explant after inoculation in step S3 into a sterilized culture medium for culture to induce hairy roots; the culture medium includes vermiculite and soil.

[0012] In some embodiments of the present invention, the Agrobacterium rhizogenes mentioned in step S2 includes Agrobacterium rhizogenes K599.

[0013] In some embodiments of the present invention, the Agrobacterium rhizogenes carries PMDC-35Spro::RUBY Plasmid.

[0014] In some embodiments of the present invention, the method for preparing the Agrobacterium rhizogenes infection solution in step S2 includes:

[0015] Add Agrobacterium rhizogenes bacterial culture to TY liquid medium and incubate until the culture system reaches OD. 600 The concentration was 0.6-1, and then 25-35 mg / L of acetylsuccinone was added to the culture system. The culture was then carried out in a shaker at 26℃-30℃ for 1-3 h to obtain the Agrobacterium rhizogenes infection solution.

[0016] In some embodiments of the present invention, the method for preparing Agrobacterium rhizogenes cells in step S2 includes:

[0017] Agrobacterium rhizogenes bacterial suspension was spread onto TY solid medium and cultured at 26℃~30℃ for 16~24 h to obtain Agrobacterium rhizogenes cells.

[0018] In some embodiments of the present invention, the method for preparing the Agrobacterium rhizogenes bacterial solution includes:

[0019] The K599 competent bacterial culture was melted on ice, and then added... PMDC-35Spro::RUBY Plasmids were placed on ice for 3-7 min; the bacterial culture was then flash-frozen in liquid nitrogen for 3-7 min, then incubated in a 37°C water bath for 3-7 min, and then placed on ice for 1-3 min. Antibiotic-free TY liquid medium was added, and the culture was incubated on a shaker at 28°C for 1-3 h. The culture was then spread onto TY solid medium (containing 45-55 μg / mL kanamycin and 45-55 μg / mL streptomycin) and incubated upside down in a 28°C incubator for 1-3 days. Single colonies from the bacterial plate were picked and transferred to 150-250 μL of TY liquid medium (containing 45-55 μg / mL kanamycin and 45-55 μg / mL streptomycin) and incubated overnight on a shaker at 28°C to obtain Agrobacterium rhizogenes bacterial culture.

[0020] In some embodiments of the present invention, the mixing ratio of vermiculite and soil in step S4 is (2~4):1.

[0021] In some embodiments of the invention, the soil includes peat soil.

[0022] In some embodiments of the present invention, the light conditions for cultivation in step S4 are as follows: first, dark cultivation for 20-28 hours, followed by 14-18 hours of light cultivation and 6-10 hours of dark cultivation per day.

[0023] In some embodiments of the present invention, the culture time in step S4 is 45 to 90 days.

[0024] In some embodiments of the present invention, the culture time in step S4 is 60 to 80 days.

[0025] In some embodiments of the present invention, the humidity of the culture environment in step S4 is 70% to 80%.

[0026] In some embodiments of the present invention, the temperature of the culture environment in step S4 is 20~25°C.

[0027] According to a second aspect of the present invention, the method for inducing transgenic hairy roots of camphor tree as described in the first aspect of the present invention is proposed for application in the expansion culture of hairy roots.

[0028] According to a third aspect of the present invention, the method for inducing transgenic hairy roots of camphor tree as described in the first aspect of the present invention is proposed for use in the preparation of natural borneol.

[0029] The present invention has at least the following beneficial effects:

[0030] This invention innovatively uses fresh leaves of a specific age as explants, achieving a 100% induction rate of hairy roots in *Camphora flavescens* and a transgenic positivity rate of >80% in an open environment. The establishment of this technical system overcomes the technical bottleneck of genetic transformation of *Camphora flavescens*, providing technical support for further targeted cultivation of high-quality seedlings through molecular modification. Furthermore, the resulting hairy roots are rich in natural borneol, providing a pathway for obtaining natural borneol resources through the further development and optimization of hairy root propagation. Attached Figure Description

[0031] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0032] Figure 1 This is a schematic diagram of the selection of explants in Embodiment 1 of the present invention; wherein the scale bar is 2 cm.

[0033] Figure 2 In Embodiment 1 of the present invention PMDC-35Spro::Ruby A schematic diagram of a plasmid;

[0034] Figure 3 This is an image showing the initial stage of leaf and bacterial co-culture in Example 1 of the present invention.

[0035] Figure 4 This is an external image of the callus tissue generated by leaf dedifferentiation in Example 1 of the present invention;

[0036] Figure 5 This is an external view of the hairy roots generated at the leaf base in Embodiment 1 of the present invention; the scale bar is 1 cm.

[0037] Figure 6 This is a diagram showing the PCR detection and identification results of transgenic hairy roots in Example 1 of the present invention;

[0038] Figure 7 The image shows the gas chromatographic detection results of the content of dextrorotatory borneol (natural borneol) in hairy roots in Example 1 of the present invention; wherein, (a) is a standard curve drawn using borneol standard, and (b) is a chromatogram of the hairy root sample to be tested and the borneol standard.

[0039] Figure 8 The following are observation results of Example 2 of the present invention after 60 days of culture; wherein, (a) shows the state of the explant inserted into the solid culture medium, scale bar: 1 cm; (b) and (c) show the appearance of the leaves after 60 days of culture, after being inserted into the solid culture medium; (d) and (e) show the appearance of the leaf base after 60 days of culture, corresponding to the red box positions in (b) and (c) respectively; (f) shows the state of the explant after 60 days of culture, with the explant laid flat in the solid culture medium; the scale bar is 1 cm in all cases.

[0040] Figure 9The images show the observation results of Example 3 of the present invention after 60 days and 90 days of cultivation; where (a) and (b) are the appearance of two leaves adjacent to the apical bud after cultivation, and (c) and (d) are the appearance of the sixth and seventh leaves below the apical bud after cultivation. The scale bar is 1 cm.

[0041] Figure 10 The images shown are the appearance of the branches during the cultivation process and the PCR verification results of Example 4 of the present invention; where (a) to (e) are the appearance of the branches at different cultivation stages, and (f) is the PCR identification result of the hairy roots. Detailed Implementation

[0042] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0043] Unless otherwise specified, all instruments and reagents used in the following examples are commercially available instruments and reagents that were purchased through conventional channels.

[0044] Example 1

[0045] This embodiment provides a method for inducing transgenic hairy roots of camphor tree, the specific steps of which are as follows:

[0046] 1) Explant preparation: Leaves were collected from one-year-old camphor trees. The third to fourth leaves below the terminal bud were selected, showing thick texture, little cuticle, and a darker color. The collected leaves should look like... Figure 1 As shown in numbers 3 and 4.

[0047] 2) PMDC-35Spro::RUBY Plasmids (plasmid maps as shown) Figure 2 (As shown) Introducing Agrobacterium rhizogenes K599 culture: K599 competent bacterial culture was thawed on ice, then 1 μL of [unclear text - likely a continuation of the previous sentence] was added. PMDC-35Spro::RUBY Plasmids were placed on ice for 5 min; then the bacterial culture was flash-frozen in liquid nitrogen for 5 min, incubated in a 37°C water bath for 5 min, and placed on ice for 2 min. 500 μL of antibiotic-free TY liquid medium was added, and the culture was incubated on a shaker at 28°C for 2 h. After that, the culture was spread onto TY solid medium (containing 50 μg / mL kanamycin and 50 μg / mL streptomycin) and incubated upside down in an incubator at 28°C for 2 days. Single clones from the bacterial plate were picked and transferred to 200 μL of TY liquid medium (containing 50 μg / mL kanamycin and 50 μg / mL streptomycin) and incubated overnight on a shaker at 28°C. PCR identification was then performed, and positive clones were preserved.

[0048] 3) Preparation of bacterial culture: Take the bacterial culture obtained in step 2) PMDC-35spro::RUBY 1 mL of Agrobacterium rhizogenes K599 bacterial culture containing plasmid was added to 100 mL of TY liquid medium containing antibiotics (50 μg / mL kanamycin and 50 μg / mL streptomycin) and cultured overnight until the bacterial culture reached OD. 600 At approximately 0.8, add 30 mg / L acetylsuccinone (AS) to the overnight cultured bacterial solution, and incubate at 28°C on a shaker for 2 h to obtain the infecting bacterial solution; simultaneously, take 200 μL of the aforementioned OD 600 The bacterial culture at a concentration of 0.8 was spread onto TY solid medium (containing 50 μg / mL kanamycin + 50 μg / mL streptomycin + 30 mg / L AS) and incubated overnight at 28°C.

[0049] 4) Explant infection: In the bacterial culture obtained in step 3), cut the petiole of the leaf collected in step 1) at the intersection of the three veins at the leaf base, exposing the leaf base. Apply the bacterial cells cultured overnight in the solid culture medium in step 3) to the leaf base, ensuring the bacterial cells fully cover the leaf base. Figure 3 ).

[0050] 5) Hairy root induction: In an open environment, the leaf bases of the explants inoculated in step 4) were inserted into sterile culture medium (vermiculite: peat moss mixed in a 3:1 volume ratio) (see [link to culture medium]). Figure 3 The cultivation environment had a humidity of approximately 75% and a temperature of 22℃. It was initially cultured in darkness for 24 hours, followed by approximately two weeks of cultivation in a greenhouse with 16 hours of light and 8 hours of darkness daily. Under the influence of Agrobacterium rhizogenes, the leaves dedifferentiated to produce callus tissue. Figure 4 After about 7 weeks of cultivation, hairy roots will appear at the main vein of the leaf. Figure 5 (This shows the hairy root growth of the two groups of leaves).

[0051] The generated single hairy roots were collected and numbered, and then genomic DNA was extracted. Using the extracted DNA as a template, PCR amplification was performed using primers specific to the ruby ​​gene. The electrophoresis results clearly show specific bands amplified in lanes 1-21. Among them, strains 1-3 are three leaves from the same *Cinnamomum camphora* plant, and lane 1 is... PMDC-35spro::RUBY Plasmids served as positive controls. Lanes 2-21 contained single hairy root samples from three leaves, while lanes 22 and 23 contained genomic DNA from roots of uninfected plants and ddH2O, respectively, serving as negative controls. The sample groups amplified bands identical to the positive controls, while the negative controls did not amplify any specific bands (e.g., plasmids were used as positive controls). Figure 6 (As shown in Table 1). The positive rate of transgenic hairy roots produced by this method is >80%, and the statistical results of the transgenic positive rate are shown in Table 1.

[0052] Table 1. Statistical results of transgenic positivity rate

[0053]

[0054] In this embodiment, after more than three repeated experiments, hairy roots were stably generated at the leaf base of leaves cultured for 60 days. A randomly selected hairy root sample was flash-frozen in liquid nitrogen, ground into powder, and transferred to a centrifuge tube containing 400 μL of anhydrous ethanol. The powder was vortexed and soaked for 48 h, followed by rotary evaporation to remove the ethanol. The precipitate was dissolved in ethyl acetate for identification and content determination of natural borneol. The gas chromatography detection program was: Column: TG-5MS; Front Inlet: 230℃; Back Detector: 260℃; Oven: 45℃→90℃ (10℃ / min, 90℃ 1 min); 90℃→200℃ (20℃ / min, 200℃ 3 min). The hairy roots induced in this embodiment contained natural borneol, with a content of approximately 1.67 mg / kg. Figure 7 ). Figure 7 In the figure, (a) is the standard curve plotted using borneol standard, and (b) is the chromatogram of the hairy root sample to be tested and the borneol standard.

[0055] Example 2

[0056] This embodiment provides a method for inducing transgenic hairy roots of camphor tree, the specific steps of which are as follows:

[0057] 1) Explant preparation: Leaves were collected from one-year-old camphor trees. The third to fourth leaves under the terminal bud were selected, which were thick, had little cuticle, and were dark in color. They were first washed three times with sterile water in a clean bench, and then soaked in 0.1% mercuric chloride solution for 8 minutes for disinfection. After disinfection, they were washed five times with sterile water for use.

[0058] 2) The plasmid transfection, bacterial culture preparation, and explant infection procedures are completely consistent with steps 2) to 4) in Example 1.

[0059] 3) Hairy root induction: Insert the leaf base of the inoculated explant into a clean bench (see...). Figure 8 (a) or tiling (see also) Figure 8 (f) was added to sterilized 1 / 2 MS solid medium and cultured in the dark for 24 h, followed by incubation at 22℃ with a photoperiod of 16 h light and 8 h dark. The leaf appearance before the start of culture was as follows. Figure 8 As shown in (b) and (c) in the figure.

[0060] In this embodiment, after more than three repeated experiments, no hairy roots were observed at the leaf base of the leaves cultured for 60 days. Magnified observation under a stereomicroscope revealed that most cells at the leaf base had died (see...). Figure 8 (d) and (e) in the text.

[0061] Example 3

[0062] This embodiment provides a method for inducing transgenic hairy roots of camphor tree, the specific steps of which are as follows:

[0063] 1) Explant preparation: Leaves were collected from a one-year-old camphor tree, selecting two leaves adjacent to the terminal bud (see appearance). Figure 1 (numbered 1 and 2 in the middle), and the sixth and seventh sequential blades below (see appearance) Figure 1 Numbers 6 and 7 in the list are reserved.

[0064] 2) Plasmid transfection, bacterial culture preparation, explant infection operation, and hairy root induction are completely consistent with steps 2) to 5) in Example 1.

[0065] In this embodiment, after more than three repeated experiments, no hairy roots were observed at the leaf base of the leaves cultured for 60 days. The selected explant leaves were not of suitable age, resulting in the failure to develop hairy roots. If the leaves are too young, they will turn black and wither during the culture process. Figure 9 As shown in (a) and (b); if the leaves are too old, they will not develop hairy roots even after 90 days of cultivation, and the base of the leaves will have rotted due to prolonged immersion in vermiculite, as... Figure 9 As shown in (c) and (d) in the figure.

[0066] Example 4

[0067] This embodiment provides a method for inducing transgenic hairy roots of camphor tree, the specific steps of which are as follows:

[0068] 1) Explant preparation: Select one-year-old camphor tree branches (0.5~1 cm in diameter), and use a sharp knife to peel off the bark (including periderm, primary phloem and secondary phloem) at the upper middle part of the branch to expose the xylem for later use.

[0069] 2) Plasmid transfection: Completely consistent with step 2) in Example 1.

[0070] 3) Preparation of bacterial solution: Take the sample containing... PMDC-35spro::RUBY 1 mL of Agrobacterium rhizogenes K599 bacterial culture with plasmid was added to 50 mL of TY liquid medium containing a specific antibiotic and cultured overnight until the bacterial culture reached OD. 600 = 0.8~1.2, centrifuge at 5000× g for 10 min at room temperature, and dilute the bacterial suspension to OD after centrifugation.600 Around 0.6 (resuspension: 1 / 4 MS medium + 0.1% sucrose + 100 μM AS + 0.015% Silwet-77, pH=5.6).

[0071] 4) Explant inoculation: Ensure the surface of the branch wound is clean. Use a sterile brush to apply the resuspended bacterial solution from step 3) to the branch wound. After application, seal the wound with plastic wrap and cover it with aluminum foil to block light. Incubate for 10-15 weeks until hairy roots appear. Figure 10 In (a)~(e)), four hairy roots were randomly selected as representative samples, genomic DNA was extracted, and PCR amplification was performed. The "+" lanes in the electrophoresis diagram represent... PMDC-35spro::RUBY The plasmid served as a positive control, lanes "1-4" represented four hairy root samples, and lanes "-" represented genomic DNA from roots of uninfected plants as a negative control. Samples in lanes 1-4 amplified bands identical to the positive control, while the negative control group did not amplify any specific bands. Figure 10 (f)). PCR identification showed that all the hairy roots in the samples were transgenic positive roots.

[0072] Both this embodiment and Example 1 successfully induced transgenic hairy roots. However, the explant used in this embodiment was a stem segment of camphor tree, which has a limited source of material and causes greater damage to the plant. In addition, the time required to induce hairy roots in this embodiment was longer, resulting in a smaller number of hairy roots. The statistical results of the number of hairy roots induced in Examples 1 and 4 are shown in Table 2. Therefore, Example 1 is a better approach.

[0073] Table 2. Statistics on the number of hairy roots induced

[0074]

[0075] This invention provides a method for inducing transgenic hairy roots of Camphor tree. Using Agrobacterium rhizogenes K599 as the experimental strain, an experiment was designed to explore the optimal conditions for hairy root regeneration of Camphor tree based on two factors: selection of infected organs / tissues and post-infection culture methods. The optimal conditions obtained through this exploration were used to screen and develop a high-rooting-rate technology system. Specifically: using fresh Camphor tree leaves as explants, the petiole was cut at the junction of the three veins at the leaf base, and Agrobacterium rhizogenes cells (K599) were placed on a specific inoculum plate. PMDC-35spro::RUBYThe bacteria were applied to the wound at the base of the leaf. After the procedure, the leaf containing the bacteria was inserted into a sterilized medium (vermiculite:soil = 3:1). After 24 hours of dark incubation, transgenic hairy roots were produced at the leaf base after approximately 60 days of cultivation in a greenhouse. Vermiculite leaf cuttings utilize the interaction between the leaf and the natural ecological environment. The cultivation conditions are simple and the operation is easy to perform. PCR testing showed that the transgenic hairy root induction rate was >80%. This technology effectively overcomes the technical barriers to establishing a transgenic camphor tree system. The cultivation method is simple, using pure vermiculite medium and watering, resulting in low cost.

[0076] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A method for inducing transgenic hairy roots of camphor tree, characterized in that, The induction method includes the following steps: S1: Collect the third to fourth leaves below the terminal bud of camphor tree as explants; S2: Preparation of Agrobacterium rhizogenes infection solution and Agrobacterium rhizogenes cells; The method for preparing the Agrobacterium rhizogenes cells includes: Agrobacterium rhizogenes bacterial suspension was spread onto TY solid medium and cultured at 26℃~30℃ for 16~24 h to obtain Agrobacterium rhizogenes bacterial cells; S3: Place the leaves collected in step S1 into the Agrobacterium rhizogenes infection solution obtained in step S2, cut off the petiole to expose the leaf base, and apply the Agrobacterium rhizogenes bacteria obtained in step S2 to the leaf base to obtain the inoculated explant leaf base. S4: Insert the leaf base of the explant after inoculation in step S3 into a sterilized culture medium for cultivation to induce hairy roots; the culture medium is vermiculite and soil; the mixing ratio of vermiculite and soil is (2~4):

1.

2. The induction method according to claim 1, characterized in that, The Agrobacterium rhizogenes mentioned in step S2 includes Agrobacterium rhizogenes K599.

3. The induction method according to claim 2, characterized in that, The Agrobacterium rhizogenes carries PMDC-35Spro:: RUBY Plasmid.

4. The induction method according to claim 1, characterized in that, The method for preparing the Agrobacterium rhizogenes infection solution in step S2 includes: Add Agrobacterium rhizogenes bacterial culture to TY liquid medium and incubate until the culture system reaches OD. 600 The concentration was 0.6-1, and then 25-35 mg / L of acetylsuccinone was added to the culture system. The culture was then carried out in a shaker at 26℃-30℃ for 1-3 h to obtain the Agrobacterium rhizogenes infection solution.

5. The induction method according to claim 1, characterized in that, The light conditions for cultivation in step S4 are as follows: first, cultivate in the dark for 20-28 hours, then cultivate in the light for 14-18 hours and in the dark for 6-10 hours each day.

6. The induction method according to claim 1, characterized in that, The culture time described in step S4 is 45 to 90 days.

7. The method for inducing transgenic hairy roots of camphor tree according to any one of claims 1 to 6 is applied to the expansion culture of hairy roots.

8. The method for inducing transgenic hairy roots of camphor tree according to any one of claims 1 to 6 in the preparation of natural borneol.

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