Cinnamomum camphora adventitious root suspension culture and d-borneol efficient accumulation method

Abstract

The invention discloses a method for cinnamomum camphora adventitious root suspension culture and efficient d-borneol accumulation, and belongs to the technical field of plant tissue culture. The invention discloses a method for cinnamomum camphora adventitious root suspension culture and efficient d-borneol accumulation, which comprises the following steps: establishing a cinnamomum camphora sterile regeneration system to obtain sterile seedlings, inducing the sterile seedlings to generate adventitious roots, preparing a plant growth-promoting rhizobacteria PGPR extracting solution, and culturing and domesticating the adventitious roots by using a suspension culture medium containing the PGPR extracting solution. The domesticated adventitious roots are subjected to efficient d-borneol accumulation culture in an induction culture medium, and the accumulated d-borneol is extracted and detected. The method effectively solves the problems of long production period, low yield and unstable components of the traditional method, and provides reliable technical support for realizing large-scale and green production of the d-borneol.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and more specifically to a method for suspension culture of adventitious roots of camphor tree and efficient accumulation of dextrorotatory borneol. Background Technology

[0002] Camphor tree (Cinnamomum camphora) is an evergreen tree belonging to the genus Cinnamomum in the family Lauraceae. It contains abundant dextrorotatory borneol. Borneol is an important natural spice and medicinal ingredient, possessing effects such as refreshing the mind, clearing heat, and relieving pain. It has wide applications in medicine, cosmetics, and fragrances. The traditional method of obtaining borneol mainly involves felling camphor trees and extracting borneol from their trunks and branches. This method not only damages the ecological environment but also results in low yields and high costs, failing to meet market demand.

[0003] With the development of plant tissue culture technology, obtaining borneol by culturing the adventitious roots of camphor tree has become a new approach. Plant adventitious root suspension culture technology, as a highly efficient plant tissue culture method, has advantages such as rapid growth, strong accumulation capacity of secondary metabolites, artificially controllable culture conditions, and ease of large-scale production, providing an effective way to solve the problem of shortage of rare medicinal plant resources.

[0004] However, the current technology for suspension culture of adventitious roots of Camphor Tree is not yet mature, and problems such as slow growth rate, low biomass, and unstable content of effective components exist. Therefore, optimizing the suspension culture method of Camphor Tree adventitious roots to improve their growth performance and the content of effective components is of significant practical importance.

[0005] Therefore, providing a method for the suspension culture of adventitious roots of camphor tree and the efficient accumulation of dextrorotatory borneol is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a method for suspension culture of camphor tree adventitious roots and efficient accumulation of dextrorotatory borneol. This method is a stable, scale-up, short-cycle, high-content dextrorotatory borneol, and highly reproducible method for suspension culture and directional induction of camphor tree adventitious roots, enabling large-scale and green production of dextrorotatory borneol.

[0007] This invention first establishes a sterile regeneration system for camphor trees to obtain sterile seedlings, which are then induced to produce adventitious roots. The core of this method lies in using plant rhizosphere growth-promoting bacteria (PGPR) extract, combined with a segmented liquid suspension culture and acclimatization process, and utilizing an optimized induction medium to significantly promote the rapid growth of adventitious roots and the efficient synthesis of dextrorotatory borneol. This invention effectively solves the problems of long production cycles, low yields, and unstable composition associated with traditional methods, providing reliable technical support for the large-scale, green production of dextrorotatory borneol.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A method for the suspension culture of adventitious roots of Camphor Tree and the efficient accumulation of dextrorotatory borneol includes the following steps: S1. Establish a camphor camphor aseptic regeneration system to obtain aseptic seedlings: Take the terminal stem segments of healthy camphor tree branches, rinse with running water for 30–60 min, rinse 3–5 times with sterile water under sterile conditions, and blot dry with sterile absorbent paper. Soak in 75% alcohol for 30–60 s, rinse 3–5 times with sterile water, then treat with 0.1 g / L mercuric chloride solution for 8–10 min, rinse 3–5 times with sterile water, and then inoculate into solid regeneration medium. Culture at 22–26℃ and 2000–3000 Lux light for 10–14 h / d for 45–50 days to obtain sterile seedlings.

[0010] The solid regeneration medium is based on MS medium, with the addition of 25-35 g / L sucrose, 1.0-2.0 mg / L 6-BA, 0.1-0.5 mg / L NAA, 0.5-1.5 g / L activated carbon, 6-8 g / L agar, and pH controlled at 5.8-6.0.

[0011] S2. Inducing the aseptic seedlings to produce adventitious roots: Take healthy stem segments from the sterile seedlings obtained in step S1, cut them into segments with a length of 0.8-1.2 cm, place them flat in the adventitious root direct induction medium, and culture them at 23-27℃ in the dark for 18-25 days to induce the production of adventitious roots.

[0012] The adventitious root direct induction medium was based on 1 / 2 MS basal medium, with the addition of 25-35 g / L sucrose, 0.5-1.5 mg / L IBA, 0.1-0.3 mg / L NAA, 0.1-0.3 mg / L KT, and 0.5-1.5 g / L PVP, and the pH was controlled at 5.8-6.0.

[0013] S3. Preparation of plant rhizosphere growth-promoting bacteria PGPR extract: strain Bacillus belye ( Bacillus velezensis After activating LS69, a seed culture was prepared and transferred to sterile LB liquid medium at an inoculum rate of 1-3%. Fermentation was carried out at 26-30℃ and 150-200 rpm for 36-60 hours until the cell concentration was ≥1×10⁻⁶. 9 The fermentation broth was obtained by centrifuging the fermentation broth and collecting the supernatant. The supernatant was then filtered through a 0.22 μm filter membrane to remove bacteria and obtain sterile PGPR extract.

[0014] Bacillus belesiensis ( Bacillus velezensis For LS69, see the reference LIU GQ, KONG YY, FANY J, et al. Data on genome analysis of Bacillus velezensis LS69[J]. Data inBrief, 2017, 13: 1-5. https: / / doi.org / 10.1016 / j.dib.2017.04.053.

[0015] S4. Cultivate and acclimatize adventitious roots using a culture medium containing PGPR extract: Cut the adventitious roots obtained in step S2 into 1.0-2.0 cm root segments, inoculate them in liquid suspension culture medium, and culture them in the dark at 22-28℃ and 100-120 rpm.

[0016] The liquid suspension medium was based on 1 / 2 MS basal medium, with the following added ingredients: 25-35 g / L sucrose, 1.0-1.5 mg / L NAA, 1.5-2.5 mg / L IBA, 0.1-0.5 mg / L 6-BA, 1.5-2.5 mg / L potassium humate, 4.0-6.0 mg / L vitamin C, 0.5-1.5 mg / L β-nicotinamide mononucleotide, and 40-60 mL / L PGPR extract. The pH was controlled at 5.8-6.0.

[0017] After 35 days of culture, subculture is carried out. During subculture, the subculture suspension medium is changed every 5-9 days. Subculture is carried out 2-4 times for acclimatization.

[0018] The subculture suspension medium was based on 1 / 2 MS basal medium, with the following added ingredients: 25-35 g / L sucrose, 1.0-1.5 mg / L NAA, 1.5-2.5 mg / L IBA, 0.1-0.5 mg / L 6-BA, 1.5-2.5 mg / L potassium humate, 4.0-6.0 mg / L vitamin C, 0.5-1.5 mg / L β-nicotinamide mononucleotide, and 10-30 mL / L PGPR extract. The pH was controlled at 5.8-6.0.

[0019] S5. The domesticated adventitious roots are cultured in a culture medium for efficient accumulation of dextrorotatory borneol: The adventitious roots acclimatized in step S4 were transferred to a suspension induction medium and cultured in the dark at 22-28℃ and 110-120 rpm.

[0020] The suspension induction medium was based on 1 / 2 MS medium, with the following added ingredients: sucrose 25-35 g / L, NAA 1.0-1.5 mg / L, IBA 1.5-2.5 mg / L, 6-BA 0.1-0.5 mg / L, potassium humate 1.5-2.5 mg / L, vitamin C 4.0-6.0 mg / L, phenylalanine 30-50 mg / L, and PGPR extract 70-90 mL / L. The pH was controlled at 5.8-6.0.

[0021] S6. Extraction and detection of accumulated dextrorotatory borneol.

[0022] Further, the extraction and detection of accumulated dextrorotatory borneol in step S6 are specifically carried out as follows: the adventitious roots harvested in step S5 are dried at 45°C to constant weight, ground into powder, anhydrous ethanol is added at a material-to-liquid ratio of 1:10 (g / mL), ultrasonically extracted at 40°C for 120 min, and the supernatant is collected by centrifugation; qualitative confirmation is performed by GC-MS, and quantitative determination is performed by GC-FID external standard method.

[0023] Furthermore, the method is applied to increasing the content of dextrorotatory borneol in the adventitious roots of Camphor tree.

[0024] As can be seen from the above technical solution, compared with the prior art, this invention discloses a method for suspension culture of adventitious roots of *Cinnamomum camphora* and efficient accumulation of dextrorotatory borneol. By optimizing the selection and disinfection methods of explants, the success rate of adventitious root induction is significantly improved, laying a good foundation for subsequent culture. Based on this, a segmented liquid suspension culture technology is innovatively adopted, combined with multiple acclimatization processes to optimize the growth environment, significantly increasing the borneol content of *Cinnamomum camphora* adventitious roots and effectively improving production efficiency. The core technology lies in the precise optimization of the culture medium formula, rationally combining auxin-like substances, sucrose, activated carbon, and other components, and specifically adding specific hormones and inducing factors, which significantly promotes the rapid growth and biomass accumulation of adventitious roots, while efficiently driving the biosynthesis of dextrorotatory borneol, ultimately obtaining adventitious roots with high dextrorotatory borneol content. During the suspension culture process, precise control of temperature, light, and rotation speed, and regular replacement of the culture medium ensure that the adventitious roots are in the optimal growth environment, further guaranteeing the content of effective components. This method is simple to operate, easy to scale up, and completely eliminates dependence on natural *Cinnamomum camphora* roots, avoiding the destruction of wild resources. The entire cultivation process is free of heavy metals and pesticide residues, making it safe and controllable. It provides strong technical support for the sustainable development and utilization of camphor tree resources, enabling large-scale factory production and possessing extremely broad application prospects. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0026] Figure 1 This is a photograph of the sterile seedlings of camphor tree in the example.

[0027] Figure 2 The image shown is a photograph of the adventitious roots of Camphor Tree (Cinnamomum camphora) in the example.

[0028] Figure 3 This is a photograph of the adventitious roots of Camphor Tree in suspension culture, as shown in the example. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Unless otherwise specified, all raw materials and reagents used in this invention are from the conventional market.

[0031] Abbreviations involved: PGPR: Plant Growth-Promoting Rhizobacteria.

[0032] MS: Murashige and Skoog Medium.

[0033] 6-BA: 6-Benzylaminopurine.

[0034] NAA: Naphthaleneacetic acid (1-Naphthaleneacetic acid).

[0035] IBA: Indole-3-butyric acid.

[0036] KT: Kinetin.

[0037] 2,4-D: 2,4-Dichlorophenoxyacetic acid.

[0038] PVP: Polyvinylpyrrolidone.

[0039] Vc: Vitamin C or Ascorbic Acid.

[0040] CFU: Colony-Forming Units.

[0041] GC-MS: Gas Chromatography-Mass Spectrometry.

[0042] GC-FID: Gas Chromatography-Flame Ionization Detector.

[0043] Example 1 A method for the suspension culture of adventitious roots of Camphor Tree and the efficient accumulation of dextrorotatory borneol, specifically including the following steps: (1) Establishment of the camphor camphor sterile system Using 330 mL glass tissue culture flasks, 40 mL of prepared solid regeneration medium was added to each flask. The flasks were autoclaved at 121℃ for 20 min, and then cooled and solidified for later use. Healthy terminal stem segments of camphor tree branches were selected as explants. After rinsing with running water for 45 min, the explants were rinsed four times with sterile water under aseptic conditions and then blotted dry with sterile absorbent paper. Subsequently, they were immersed in 75% ethanol solution for 45 s, rinsed four times with sterile water, disinfected with 0.1 g / L mercuric chloride solution for 9 min, and finally rinsed four times with sterile water. The sterilized explants were inoculated into solid regeneration medium and cultured for 48 days at 24℃, light intensity of 2500 Lux, and photoperiod of 12 h / d to obtain regenerated sterile seedlings. Figure 1 ).

[0044] The solid regeneration medium contains the following components: MS medium as the base, with the addition of 30 g / L sucrose, 1.5 mg / L 6-BA, 0.3 mg / L NAA, 1.0 g / L activated carbon and 7.0 g / L agar, and a pH of 5.9.

[0045] (2) Induction of adventitious roots Using 330 mL glass tissue culture flasks, add 40 mL of prepared adventitious root direct induction medium to each flask, autoclave at 121℃ for 20 min, and let cool and solidify before use. Take healthy stem segments from the sterile seedlings obtained in step (1), cut them into 1.0 cm long segments, and lay them flat on the adventitious root direct induction medium. Inoculate 5 segments per flask, ensuring that the base of the stem segment is in full contact with the medium. Culture at 25℃ in the dark for 21 days, and adventitious roots will be induced at the cut ends of the stem segments ( Figure 2 ).

[0046] The adventitious root direct induction medium was based on 1 / 2 MS medium, with the addition of 30 g / L sucrose, 1.0 mg / L IBA, 0.2 mg / L NAA, 0.2 mg / L KT and 1.0 g / L PVP, and the pH value was 5.9.

[0047] (3) Preparation of PGPR extract Seed culture was prepared by activating strain Bacillus belye LS69, and then transferred to sterile LB liquid medium at a 2% inoculum. Fermentation was carried out at 28°C and 180 rpm for 48 hours until the cell concentration reached 1×10⁻⁶. 9 The fermentation broth was obtained by centrifuging the fermentation broth and then filtering the supernatant through a 0.22 μm filter membrane to obtain a sterile plant rhizosphere growth-promoting bacteria (PGPR) extract.

[0048] (4) Liquid culture and acclimatization of adventitious roots Use 250 mL glass Erlenmeyer flasks, add 100 mL of prepared liquid suspension culture medium to each flask, autoclave at 121℃ for 20 min, and cool for later use. Cut the adventitious roots obtained in step (2) into root segments with a length of 1.5 cm, inoculate them into liquid suspension culture medium, and culture them at 25℃, 115 rpm, and in the dark for 35 days.

[0049] The liquid suspension medium was based on 1 / 2 MS basal medium, with the following added ingredients: 30 g / L sucrose, 1.3 mg / L NAA, 2.0 mg / L IBA, 0.3 mg / L 6-BA, 2.0 mg / L potassium humate, 5.0 mg / L vitamin C, 1.0 mg / L β-nicotinamide mononucleotide, 50 mL / L PGPR extract, and pH controlled at 5.9.

[0050] The newly formed adventitious roots were transferred to a subculture suspension medium for subculture, and the medium was changed every 7 days for 3 generations.

[0051] The subculture suspension medium was based on 1 / 2 MS basal medium, with the addition of 30 g / L sucrose, 1.3 mg / L NAA, 2.0 mg / L IBA, 0.3 mg / L 6-BA, 2.0 mg / L potassium humate, 5.0 mg / L vitamin C, 1.0 mg / L β-nicotinamide mononucleotide, 20 mL / L PGPR extract, and pH controlled at 5.9.

[0052] (5) High-efficiency accumulation and cultivation of dextrorotatory borneol Use 250 mL Erlenmeyer flasks, add 100 mL of prepared suspension induction medium to each flask, autoclave at 121℃ for 20 min, and cool for later use. Transfer the acclimatized adventitious roots to the suspension induction medium and culture at 25℃, 115 rpm, and in the dark for 35 days before harvesting the adventitious roots. Figure 3 ).

[0053] The suspension induction medium was based on 1 / 2 MS basal medium, with the following added: 30 g / L sucrose, 1.3 mg / L NAA, 2.0 mg / L IBA, 0.3 mg / L 6-BA, 2.0 mg / L potassium humate, 5.0 mg / L vitamin C, 40 mg / L phenylalanine, 80 mL / L PGPR extract, and pH controlled at 5.9.

[0054] (6) Extraction and content determination of dextrorotatory borneol: The harvested adventitious roots were dried at 45℃ to constant weight and ground into powder. 0.5 g of powder was accurately weighed and added to anhydrous ethanol at a material-to-liquid ratio of 1:10 (g / mL) in two portions (2.5 mL before sonication, and another 2.5 mL after sonication for 60 min). Extraction was performed by sonication at 40℃ for 120 min, followed by centrifugation for 3 min. The supernatant was then collected for qualitative analysis using gas chromatography-mass spectrometry (GC-MS) and quantitative analysis using external standard gas chromatography (GC).

[0055] Gas chromatography conditions: DB-17 capillary column (30 m × 0.25 mm × 0.25 μm); Inlet temperature: 220 ℃; Splitless injection, splitless time: 1 min; Injection volume: 1 μL; Carrier gas (N2) column flow rate: 2 mL / min; Purge gas flow rate: 3 mL / min. Temperature program: Initial temperature 60℃, hold for 1 min, increase to 90℃ at 5 ℃ / min, then increase to 180℃ at 20 ℃ / min. Detector FID, temperature: 240 ℃, H2 flow rate: 30 mL / min, O2 flow rate: 400 mL / min, make-up flow rate: 25 mL / min.

[0056] Gas chromatography-mass spectrometry analysis conditions: Gas chromatography conditions: DB-5MS capillary column (30 m × 0.25 mm × 0.25 μm); injection port temperature: 220℃; splitless injection, splitless time: 1 min; injection volume: 1 μL; carrier gas (99.999% high-purity helium) column flow rate: 1.1 mL / min; purge gas flow rate: 3 mL / min. Temperature program: initial temperature 70℃, increased to 100℃ at 3℃ / min, then increased to 250℃ at 15℃ / min.

[0057] Mass spectrometry conditions: Electron impact (EI) source, electron energy 70 eV, ion source temperature 230 ℃, interface temperature 280 ℃. When SIM mode was selected, the scan mass numbers (m / z) were 95, 41, and 110.

[0058] The results showed that after 35 days of adventitious root growth, the fresh weight was 8.8 g, of which the content of dextrorotatory borneol was 31.0 mg / g DW. DW is dry weight.

[0059] Example 2 A method for the suspension culture of adventitious roots of Camphor Tree and the efficient accumulation of dextrorotatory borneol, specifically including the following steps: (1) Establishment of the camphor camphor sterile system Using 330 mL glass tissue culture flasks, 40 mL of prepared solid regeneration medium was added to each flask. The flasks were autoclaved at 121℃ for 20 min, and then cooled and solidified for later use. Healthy terminal stem segments of camphor tree branches were selected as explants. After rinsing with running water for 30 min, the explants were rinsed three times with sterile water under aseptic conditions and then blotted dry with sterile absorbent paper. Subsequently, they were immersed in 75% ethanol solution for 30 s, rinsed three times with sterile water, disinfected with 0.1 g / L mercuric chloride solution for 8 min, and finally rinsed three times with sterile water. The sterilized explants were inoculated into solid regeneration medium and cultured at 22℃, light intensity of 2000 Lux, and photoperiod of 10 h / d for 45 days to obtain regenerated sterile seedlings.

[0060] The solid regeneration medium contains the following components: MS medium as the base, with the addition of 25 g / L sucrose, 1.0 mg / L 6-BA, 0.1 mg / L NAA, 0.5 g / L activated carbon and 6.0 g / L agar, and a pH of 5.8.

[0061] (2) Induction of adventitious roots Using 330 mL glass tissue culture flasks, add 40 mL of prepared adventitious root direct induction medium to each flask, autoclave at 121℃ for 20 min, and let cool and solidify before use. Take healthy stem segments from the sterile seedlings obtained in step (1), cut them into 0.8 cm long segments, and lay them flat on the adventitious root direct induction medium. Inoculate 5 segments per flask, ensuring that the base of the stem segment is in full contact with the medium. Culture at 23℃ in the dark for 18 days to induce adventitious roots at the cut ends of the stem segments.

[0062] The adventitious root direct induction medium was based on 1 / 2 MS medium, with the addition of 25 g / L sucrose, 0.5 mg / L IBA, 0.1 mg / L NAA, 0.1 mg / L KT and 0.5 g / L PVP, and the pH value was 5.8.

[0063] (3) Preparation of PGPR extract Seed culture was prepared by activating strain Bacillus belye LS69, and then transferred to sterile LB liquid medium at a 1% inoculum. Fermentation was carried out at 30°C and 200 rpm for 60 hours until the cell concentration reached 1×10⁻⁶. 9 The fermentation broth was obtained by centrifuging the fermentation broth and then filtering the supernatant through a 0.22 μm filter membrane to obtain a sterile plant rhizosphere growth-promoting bacteria (PGPR) extract.

[0064] (4) Liquid culture and acclimatization of adventitious roots Use 250 mL glass Erlenmeyer flasks, add 100 mL of prepared liquid suspension culture medium to each flask, autoclave at 121℃ for 20 min, and cool for later use. Cut the adventitious roots obtained in step (2) into root segments with a length of 1.0 cm, inoculate them into liquid suspension culture medium, and culture at 22℃, 100 rpm, and in the dark for 35 days.

[0065] The liquid suspension medium was based on 1 / 2 MS basal medium, with the following added ingredients: 25 g / L sucrose, 1.0 mg / L NAA, 1.5 mg / L IBA, 0.1 mg / L 6-BA, 1.5 mg / L potassium humate, 4.0 mg / L vitamin C, 0.5 mg / L β-nicotinamide mononucleotide, 40 mL / L PGPR extract, and pH controlled at 5.8.

[0066] The newly formed adventitious roots were transferred to a subculture suspension medium for subculture, and the medium was changed every 5 days for two generations of continuous acclimatization.

[0067] The subculture suspension medium was based on 1 / 2 MS basal medium, with the following added ingredients: 25 g / L sucrose, 1.0 mg / L NAA, 1.5 mg / L IBA, 0.1 mg / L 6-BA, 1.5 mg / L potassium humate, 4.0 mg / L vitamin C, 0.5 mg / L β-nicotinamide mononucleotide, 10 mL / L PGPR extract, and pH controlled at 5.8.

[0068] (5) High-efficiency accumulation and cultivation of dextrorotatory borneol Use 250 mL Erlenmeyer flasks, add 100 mL of prepared suspension induction medium to each flask, autoclave at 121℃ for 20 min, and cool for later use. Transfer the acclimatized adventitious roots to the suspension induction medium, and incubate at 22℃, 110 rpm, and in the dark for 35 days before harvesting the adventitious roots.

[0069] The suspension induction medium was based on 1 / 2 MS basal medium, with the following added ingredients: 25 g / L sucrose, 1.0 mg / L NAA, 1.5 mg / L IBA, 0.1 mg / L 6-BA, 1.5 mg / L potassium humate, 4.0 mg / L vitamin C, 30 mg / L phenylalanine, and 70 mL / L PGPR extract. The pH was controlled at 5.8.

[0070] (6) Extraction and content determination of dextrorotatory borneol: Same as in Example 1.

[0071] The results showed that after 35 days of growth, the fresh weight of the adventitious roots was 7.2 g, of which the content of dextrorotatory borneol was 29.4 mg / g DW.

[0072] Example 3 A method for the suspension culture of adventitious roots of Camphor Tree and the efficient accumulation of dextrorotatory borneol, specifically including the following steps: (1) Establishment of the camphor camphor sterile system Using 330 mL glass tissue culture flasks, 40 mL of prepared solid regeneration medium was added to each flask. The flasks were autoclaved at 121℃ for 20 min, and then cooled and solidified for later use. Healthy terminal stem segments of camphor tree branches were selected as explants. After rinsing with running water for 60 min, the explants were rinsed five times with sterile water under aseptic conditions and then blotted dry with sterile absorbent paper. They were then immersed in 75% ethanol solution for 60 s, rinsed five times with sterile water, disinfected with 0.1 g / L mercuric chloride solution for 10 min, and finally rinsed five times with sterile water. The sterilized explants were inoculated into solid regeneration medium and cultured at 26℃, 3000 Lux light intensity, and 14 h / d photoperiod for 50 days to obtain regenerated sterile seedlings.

[0073] The solid regeneration medium contains the following components: MS medium as the base, with the addition of 35 g / L sucrose, 2.0 mg / L 6-BA, 0.5 mg / L NAA, 1.5 g / L activated carbon and 8.0 g / L agar, and a pH of 6.0.

[0074] (2) Induction of adventitious roots Using 330 mL glass tissue culture flasks, add 40 mL of prepared adventitious root direct induction medium to each flask, autoclave at 121℃ for 20 min, and let cool and solidify before use. Take healthy stem segments from the sterile seedlings obtained in step (1), cut them into 1.2 cm long segments, and lay them flat on the adventitious root direct induction medium. Inoculate 5 segments per flask, ensuring that the base of the stem segment is in full contact with the medium. Culture at 27℃ in the dark for 25 days to induce adventitious roots at the cut ends of the stem segments.

[0075] The adventitious root direct induction medium was based on 1 / 2 MS medium, with the addition of 35 g / L sucrose, 1.5 mg / L IBA, 0.3 mg / L NAA, 0.3 mg / L KT and 1.5 g / L PVP, and the pH value was 6.0.

[0076] (3) Preparation of PGPR extract Seed culture was prepared by activating strain Bacillus belye LS69, and then transferred to sterile LB liquid medium at a 3% inoculum. Fermentation was carried out at 26°C and 150 rpm for 36 hours until the cell concentration reached 1×10⁻⁶. 9 The fermentation broth was obtained by centrifuging the fermentation broth and then filtering the supernatant through a 0.22 μm filter membrane to obtain a sterile plant rhizosphere growth-promoting bacteria (PGPR) extract.

[0077] (4) Liquid culture and acclimatization of adventitious roots Use 250 mL glass Erlenmeyer flasks, add 100 mL of prepared liquid suspension culture medium to each flask, autoclave at 121℃ for 20 min, and cool for later use. Cut the adventitious roots obtained in step (2) into root segments with a length of 2.0 cm, inoculate them into liquid suspension culture medium, and culture at 28℃, 120 rpm, and in the dark for 35 days.

[0078] The liquid suspension medium was based on 1 / 2 MS basal medium, with the following added ingredients: sucrose 35 g / L, NAA 1.5 mg / L, IBA 2.5 mg / L, 6-BA 0.5 mg / L, potassium humate 2.5 mg / L, vitamin C 6.0 mg / L, β-nicotinamide mononucleotide 1.5 mg / L, PGPR extract 60 mL / L, and pH controlled at 6.0.

[0079] The newly formed adventitious roots were transferred to a subculture suspension medium for subculture, and the medium was changed every 9 days for 3 generations.

[0080] The subculture suspension medium was based on 1 / 2 MS basal medium, with the following added ingredients: sucrose 35 g / L, NAA 1.5 mg / L, IBA 2.5 mg / L, 6-BA 0.5 mg / L, potassium humate 2.5 mg / L, vitamin C 6.0 mg / L, β-nicotinamide mononucleotide 1.5 mg / L, PGPR extract 30 mL / L, and pH controlled at 6.0.

[0081] (5) High-efficiency accumulation and cultivation of dextrorotatory borneol Use 250 mL Erlenmeyer flasks, add 100 mL of prepared suspension induction medium to each flask, autoclave at 121℃ for 20 min, and cool for later use. Transfer the acclimatized adventitious roots to the suspension induction medium and culture at 28℃, 120 rpm, and in the dark for 35 days before harvesting the adventitious roots.

[0082] The suspension induction medium was based on 1 / 2 MS basal medium, with the following added: sucrose 35 g / L, NAA 1.5 mg / L, IBA 2.5 mg / L, 6-BA 0.5 mg / L, potassium humate 2.5 mg / L, vitamin C 6.0 mg / L, phenylalanine 50 mg / L, PGPR extract 90 mL / L, and pH controlled at 6.0.

[0083] (6) Extraction and content determination of dextrorotatory borneol: Same as in Example 1.

[0084] The results showed that after 35 days of growth, the fresh weight of the adventitious roots was 7.8 g, of which the content of dextrorotatory borneol was 30.4 mg / g DW.

[0085] Comparative Example 1: Adventitious root culture of Camphor Tree without PGPR extract A method for suspension culture of adventitious roots of Camphor Tree and accumulation of dextrorotatory borneol, specifically including the following steps: (1) Establishment of the camphor camphor sterile system: Same as in Example 1.

[0086] (2) Induction of adventitious roots: Same as in Example 1.

[0087] (3) Liquid culture and acclimatization of adventitious roots Use 250 mL glass Erlenmeyer flasks, add 100 mL of prepared suspension culture medium to each flask, autoclave at 121℃ for 20 min, and cool for later use. Cut the adventitious roots obtained in step (2) into root segments with a length of 1.5 cm, inoculate them into liquid suspension culture medium, and culture them at 25℃, 105 rpm, and in the dark for 35 days.

[0088] The newly formed adventitious roots were transferred to liquid suspension culture medium for subculture, with the medium being changed every 7 days, for a total of 3 generations.

[0089] The suspension medium was based on 1 / 2 MS basal medium, with the following added: sucrose 30 g / L, NAA 1.3 mg / L, IBA 2.0 mg / L, 6-BA 0.3 mg / L, potassium humate 2.0 mg / L, vitamin C 5.0 mg / L, β-nicotinamide mononucleotide 1.0 mg / L, and pH controlled at 5.9.

[0090] (4) High-efficiency accumulation and cultivation of dextrorotatory borneol Use 250 mL Erlenmeyer flasks, add 100 mL of prepared induction medium to each flask, autoclave at 121℃ for 20 min, and cool for later use. Transfer the acclimatized adventitious roots to the induction medium and culture at 25℃, 115 rpm, and in the dark for 35 days before harvesting the adventitious roots.

[0091] The induction medium was based on 1 / 2 MS basal medium, with the following added: sucrose 30 g / L, NAA 1.3 mg / L, IBA 2.0 mg / L, 6-BA 0.3 mg / L, potassium humate 2.0 mg / L, vitamin C 5.0 mg / L, phenylalanine 40 mg / L, and pH controlled at 5.9.

[0092] (5) Extraction and content determination of dextrorotatory borneol: Same as in Example 1.

[0093] The results showed that after 35 days of adventitious root growth, the fresh biomass weight was only 4.2g; the content of dextrorotatory borneol was significantly reduced to 8.7mg / g DW.

[0094] Comparative Example 2: The concentration of PGPR extract was lower than that in Example 2. A method for suspension culture of adventitious roots of Camphor Tree and accumulation of dextrorotatory borneol, specifically including the following steps: (1) Establishment of the camphor camphor sterile system: Same as in Example 2.

[0095] (2) Induction of adventitious roots: Same as in Example 2.

[0096] (3) Preparation of PGPR extract: Same as in Example 2.

[0097] (4) Liquid culture and acclimatization of adventitious roots Use 250 mL glass Erlenmeyer flasks, add 100 mL of prepared suspension culture medium to each flask, autoclave at 121℃ for 20 min, and cool for later use. Cut the adventitious roots obtained in step (2) into root segments with a length of 1.0 cm, inoculate them into suspension culture medium, and culture at 22℃, 100 rpm, and in the dark for 35 days.

[0098] The suspension medium was based on 1 / 2 MS basal medium, with the following added ingredients: 25 g / L sucrose, 1.0 mg / L NAA, 1.5 mg / L IBA, 0.1 mg / L 6-BA, 1.5 mg / L potassium humate, 4.0 mg / L vitamin C, 0.5 mg / L β-nicotinamide mononucleotide, 30 mL / L PGPR extract, and pH controlled at 5.8.

[0099] The newly formed adventitious roots were transferred to a subculture suspension medium for subculture, and the medium was changed every 5 days for two generations of continuous acclimatization.

[0100] The subculture suspension medium was based on 1 / 2 MS basal medium, with the following added ingredients: 25 g / L sucrose, 1.0 mg / L NAA, 1.5 mg / L IBA, 0.1 mg / L 6-BA, 1.5 mg / L potassium humate, 4.0 mg / L vitamin C, 0.5 mg / L β-nicotinamide mononucleotide, 10 mL / L PGPR extract, and pH controlled at 5.8.

[0101] (5) High-efficiency accumulation and cultivation of dextrorotatory borneol Use 250 mL Erlenmeyer flasks, add 100 mL of prepared induction medium to each flask, autoclave at 121℃ for 20 min, and cool for later use. Transfer the acclimatized adventitious roots to the induction medium and culture at 22℃, 110 rpm, and in the dark for 35 days before harvesting the adventitious roots.

[0102] The induction medium was based on 1 / 2 MS basal medium, with the following added ingredients: 25 g / L sucrose, 1.0 mg / L NAA, 1.5 mg / L IBA, 0.1 mg / L 6-BA, 1.5 mg / L potassium humate, 4.0 mg / L vitamin C, 30 mg / L phenylalanine, and 60 mL / L PGPR extract. The pH was controlled at 5.8.

[0103] (6) Extraction and content determination of dextrorotatory borneol: Same as in Example 1.

[0104] The results showed that after 35 days of adventitious root growth, the biomass fresh weight was 5.8 g; the final dextrorotatory borneol content decreased to 21.3 mg / g DW.

[0105] Comparative Example 3: The concentration of PGPR extract was higher than that in Example 3. A method for suspension culture of adventitious roots of Camphor Tree and accumulation of dextrorotatory borneol, specifically including the following steps: (1) Establishment of the camphor camphor sterile system: Same as in Example 3.

[0106] (2) Induction of adventitious roots: Same as in Example 3.

[0107] (3) Preparation of PGPR extract: Same as in Example 3.

[0108] (4) Liquid culture and acclimatization of adventitious roots Use 250 mL glass Erlenmeyer flasks, add 100 mL of prepared suspension culture medium to each flask, autoclave at 121℃ for 20 min, and cool for later use. Cut the adventitious roots obtained in step (2) into root segments with a length of 2.0 cm, inoculate them into suspension culture medium, and culture at 28℃, 120 rpm, and in the dark for 35 days.

[0109] The suspension medium was based on 1 / 2 MS basal medium, with the following added ingredients: sucrose 35 g / L, NAA 1.5 mg / L, IBA 2.5 mg / L, 6-BA 0.5 mg / L, potassium humate 2.5 mg / L, vitamin C 6.0 mg / L, β-nicotinamide mononucleotide 1.5 mg / L, PGPR extract 70 mL / L, and pH controlled at 6.0.

[0110] The newly formed adventitious roots were transferred to a subculture suspension medium for subculture, and the medium was changed every 9 days for 3 generations.

[0111] The subculture suspension medium was based on 1 / 2 MS basal medium, with the following added ingredients: sucrose 35 g / L, NAA 1.5 mg / L, IBA 2.5 mg / L, 6-BA 0.5 mg / L, potassium humate 2.5 mg / L, vitamin C 6.0 mg / L, β-nicotinamide mononucleotide 1.5 mg / L, PGPR extract 50 mL / L, and pH controlled at 6.0.

[0112] (5) High-efficiency accumulation and cultivation of dextrorotatory borneol Use 250 mL Erlenmeyer flasks, add 100 mL of prepared induction medium to each flask, autoclave at 121℃ for 20 min, and cool for later use. Transfer the acclimatized adventitious roots to the induction medium and culture at 28℃, 120 rpm, and in the dark for 35 days before harvesting the adventitious roots.

[0113] The induction medium was based on 1 / 2 MS basal medium, with the following added ingredients: sucrose 35 g / L, NAA 1.5 mg / L, IBA 2.5 mg / L, 6-BA 0.5 mg / L, potassium humate 2.5 mg / L, vitamin C 6.0 mg / L, phenylalanine 50 mg / L, PGPR extract 100 mL / L, and pH controlled at 6.0.

[0114] (6) Extraction and content determination of dextrorotatory borneol: Same as in Example 1.

[0115] The results showed that high concentration of PGPR extract inhibited adventitious root growth, and after 35 days of adventitious root growth, the final fresh weight decreased to 6.3g; the content of dextrorotatory borneol did not increase, and was only 21.9 mg / g DW.

[0116] Comparative Example 4: No adventitious root acclimatization treatment was performed. A method for suspension culture of adventitious roots of Camphor Tree and accumulation of dextrorotatory borneol, specifically including the following steps: (1) Establishment of the camphor camphor sterile system: Same as in Example 1.

[0117] (2) Induction of adventitious roots: Same as in Example 1.

[0118] (3) Preparation of PGPR extract: Same as in Example 1.

[0119] (4) Omit the adventitious root acclimatization step and directly carry out efficient accumulation culture. Use 250 mL glass Erlenmeyer flasks, add 100 mL of prepared suspension induction medium to each flask, autoclave at 121℃ for 20 min, and cool for later use. Cut the adventitious roots obtained in step (2) into root segments with a length of 1.0 cm and transfer them to the suspension induction medium. After culturing at 25℃, rotating at 115 rpm and in the dark for 35 days, the adventitious roots are harvested.

[0120] The suspension induction medium was based on 1 / 2 MS basal medium, with the following added: sucrose 30 g / L, NAA 1.3 mg / L, IBA 2.0 mg / L, 6-BA 0.3 mg / L, potassium humate 2.0 mg / L, vitamin C 5.0 mg / L, phenylalanine 40 mg / L, PGPR extract 80 mL / L, and pH controlled at 5.9.

[0121] (5) Extraction and content determination of dextrorotatory borneol: Same as in Example 1.

[0122] The results showed that the adventitious roots were in poor condition, and after 35 days of growth, the final fresh weight was only 3.7g; the content of dextrorotatory borneol dropped significantly to 12.5 mg / g DW.

[0123] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for the suspension culture of adventitious roots of Camphor Tree and the efficient accumulation of dextrorotatory borneol, characterized in that, Includes the following steps: S1. Establish a camphor camphor aseptic regeneration system to obtain aseptic seedlings: Take the terminal stem segments of healthy camphor tree branches, rinse with running water for 30–60 min, rinse 3–5 times with sterile water under sterile conditions, and blot dry with sterile absorbent paper. Soak in 75% alcohol for 30–60 s, rinse 3–5 times with sterile water, then treat with 0.1 g / L mercuric chloride solution for 8–10 min, rinse 3–5 times with sterile water, and then inoculate into solid regeneration medium. Culture at 22–26℃ and 2000–3000 Lux light for 10–14 h / d for 45–50 days to obtain sterile seedlings. The solid regeneration medium is based on MS medium, with the addition of 25-35 g / L sucrose, 1.0-2.0 mg / L 6-BA, 0.1-0.5 mg / L NAA, 0.5-1.5 g / L activated carbon, 6-8 g / L agar, and pH controlled at 5.8-6.

0. S2. Inducing the aseptic seedlings to produce adventitious roots: Take healthy stem segments from the sterile seedlings obtained in step S1, cut them into segments with a length of 0.8-1.2 cm, place them flat in the adventitious root direct induction medium, and culture them at 23-27℃ in the dark for 18-25 days to induce the production of adventitious roots; The adventitious root direct induction medium was based on 1 / 2 MS basal medium, with the addition of 25-35 g / L sucrose, 0.5-1.5 mg / L IBA, 0.1-0.3 mg / L NAA, 0.1-0.3 mg / L KT, and 0.5-1.5 g / L PVP, and the pH was controlled at 5.8-6.

0. S3. Preparation of plant rhizosphere growth-promoting bacteria PGPR extract: strain Bacillus belye ( Bacillus velezensis After activating LS69, a seed culture was prepared and transferred to sterile LB liquid medium at an inoculum rate of 1-3%. Fermentation was carried out at 26-30℃ and 150-200 rpm for 36-60 hours until the cell concentration was ≥1×10⁻⁶. 9 CFU / mL was used to obtain the fermentation broth; after centrifugation, the supernatant of the fermentation broth was collected and then filtered through a 0.22 μm filter membrane to remove bacteria, thus obtaining a sterile PGPR extract. S4. Cultivate and acclimatize adventitious roots using a culture medium containing PGPR extract: Cut the adventitious roots obtained in step S2 into 1.0-2.0 cm root segments, inoculate them in liquid suspension culture medium, and culture them in the dark at 22-28℃ and 100-120 rpm. The liquid suspension medium was based on 1 / 2 MS basal medium, supplemented with 25-35 g / L sucrose, 1.0-1.5 mg / L NAA, 1.5-2.5 mg / L IBA, 0.1-0.5 mg / L 6-BA, 1.5-2.5 mg / L potassium humate, 4.0-6.0 mg / L vitamin C, 0.5-1.5 mg / L β-nicotinamide mononucleotide, and 40-60 mL / L PGPR extract, with the pH controlled at 5.8-6.

0. After 35 days of culture, subculture was carried out. During subculture, the subculture suspension medium was changed every 5-9 days. Subculture was carried out 2-4 times for acclimatization. The subculture suspension medium was based on 1 / 2 MS basal medium, supplemented with 25-35 g / L sucrose, 1.0-1.5 mg / L NAA, 1.5-2.5 mg / L IBA, 0.1-0.5 mg / L 6-BA, 1.5-2.5 mg / L potassium humate, 4.0-6.0 mg / L vitamin C, 0.5-1.5 mg / L β-nicotinamide mononucleotide, and 10-30 mL / L PGPR extract, with the pH controlled at 5.8-6.

0. S5. The domesticated adventitious roots are cultured in a culture medium for efficient accumulation of dextrorotatory borneol: The adventitious roots acclimatized in step S4 were transferred to a suspension induction medium and cultured in the dark at 22-28℃ and 110-120 rpm. The suspension induction medium was based on 1 / 2 MS medium, with the following added components: sucrose 25-35 g / L, NAA 1.0-1.5 mg / L, IBA 1.5-2.5 mg / L, 6-BA 0.1-0.5 mg / L, potassium humate 1.5-2.5 mg / L, vitamin C 4.0-6.0 mg / L, phenylalanine 30-50 mg / L, PGPR extract 70-90 mL / L, and pH controlled at 5.8-6.

0. S6. Extraction and detection of accumulated dextrorotatory borneol.

2. The method for suspension culture of adventitious roots of camphor tree and efficient accumulation of dextrorotatory borneol according to claim 1, characterized in that, The specific steps for extracting and detecting the accumulated dextrorotatory borneol in step S6 are as follows: the adventitious roots harvested in step S5 are dried at 45°C to constant weight, ground into powder, and anhydrous ethanol is added at a material-to-liquid ratio of 1:10 (g / mL). The mixture is then ultrasonically extracted at 40°C for 120 min, and the supernatant is collected by centrifugation. Qualitative confirmation is performed using GC-MS, and quantitative determination is performed using the GC-FID external standard method.

3. The application of the method according to claim 1 or 2 in increasing the content of dextrorotatory borneol in the adventitious roots of camphor tree.

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