Method for inducing industrial cannabis sativa adventitious roots to generate flavone and phenolic substances
By inducing industrial hemp explants with endophyte RF-1, a stable indefinite root liquid culture system was established, which solved the problem of the initial stage and low metabolites content of industrial hemp in the prior art, and achieved rapid growth of indefinite roots and significant increase in metabolites.
Patent Information
- Application Number
- CN202510474572.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-15
- Publication Date
- 2025-08-12
AI Technical Summary
The existing industrial hemp induced uncertain root production system is still in its initial stage, and the metabolites produced are not high, making it difficult to effectively induce the production of flavonoids, phenolic substances and CBD.
The endophyte RF-1 (Redopodospores) was used to induce industrial hemp explants. By inducing indefinite roots and liquid culture in solid and liquid culture medium, hormone combination and culture conditions were optimized, and combined with endophyte inducer treatment at different concentrations, a stable indefinite root liquid culture system was established.
It has achieved rapid and efficient acquisition of uncertain roots from industrial hemp explants, with good growth status, and significantly increased the content of total CBD, total flavonoids and total phenolic substances.
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Abstract
Description
Technical Field
[0001] The invention relates to a method for inducing industrial hemp adventitious roots to produce flavonoids and phenolic substances, and belongs to the field of biotechnology. Background Art
[0002] Industrial hemp (Cannabis sativa L.), also known as marijuana or hemp, is an annual herb. Extraction and identification have revealed over 588 bioactive compounds, primarily cannabinoids, flavonoids, phenols, terpenes, alkaloids, and other secondary metabolites. These metabolites exhibit analgesic, anti-inflammatory, and antibacterial activities.
[0003] Flavonoids and phenolic compounds possess excellent antioxidant, anti-inflammatory, antibacterial, and anticancer properties, and are widely used to prevent and treat cardiovascular and cerebrovascular diseases, improve blood vessel permeability, lower blood lipids, and effectively prevent hypertension and angina pectoris. Cannabidiol (CBD), a major component of cannabinoids, holds broad promise because it is non-addictive and possesses significant medicinal value in antidepressant, anti-inflammatory, and pain relief.
[0004] Plant tissue culture technology is often a process of inoculating under sterile conditions and artificially controlling the culture conditions to allow it to grow, differentiate, and proliferate. Adventitious roots formed by hormone induction or external stimulation have a shorter growth cycle, a faster proliferation rate, and can stabilize the genetic activity characteristics of plants by culturing in solid and liquid culture media. The existing industrial hemp induction adventitious root production system is still in its early stages, and the content of metabolites produced is not high. Therefore, this article uses hormones to effectively induce the production of adventitious roots from industrial hemp explants, optimizes the liquid culture conditions of various growth hormones, optimizes the effects of different concentrations of endophytic bacteria induction treatment on the physiological and biochemical state of adventitious roots and the content of metabolites, establishes a system that effectively induces the production of industrial hemp adventitious roots, and provides ideas for the rapid extraction of metabolites. Summary of the Invention
[0005] The technical problem to be solved by the present invention is to provide a method for effectively inducing industrial hemp explants to produce adventitious roots and produce flavonoids, phenolic substances and CBD, providing an efficient and rapid technical means for obtaining secondary metabolites of industrial hemp.
[0006] In order to solve the above problems, the present invention adopts the following technical means:
[0007] On the one hand, the present invention provides an endophyte isolated from industrial hemp explants, named RF-1, classified as Rhodosporidiobolus fluvialis, and deposited in the General Microbiology Center of the China Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC NO.28625 and a deposit date of October 13, 2023.
[0008] Furthermore, the present invention also proposes the use of the endophyte in inducing industrial hemp adventitious roots to produce flavonoids and phenolic substances.
[0009] Among them, preferably, the phenolic substances include cannabidiol (CBD).
[0010] In another aspect, the present invention provides a method for inducing industrial hemp adventitious roots to produce flavonoids and phenolic substances, comprising the following steps:
[0011] S1: Induction of adventitious roots of industrial hemp
[0012] The above-ground leaves of industrial hemp before flowering were selected as explants, washed and disinfected, and then inoculated into MS solid medium containing NAA and IBA to induce adventitious roots.
[0013] S2: Liquid culture of adventitious roots
[0014] Select adventitious roots with good growth status and inoculate them into liquid MS medium for liquid culture. When the adventitious roots grow to fill the bottle, remove the explant part on the adventitious roots and place them into new liquid MS medium for subculture.
[0015] S3: Preparation and induction of inducers
[0016] S3-1: inoculating the endophyte into a liquid MS culture medium and culturing the culture to obtain a fermentation broth of the endophyte RF-1; centrifuging the obtained fermentation broth of the endophyte RF-1 using an ultra-high-speed low-temperature centrifuge to separate the endophyte RF-1 cells from the culture medium, and collecting the endophyte RF-1 cells;
[0017] S3-2: The fermentation broth obtained in S3-1 and the collected endophyte RF-1 bacteria were used to prepare the following four inducers: 1) Live bacteria inducer: 0.75×10 71) endophyte RF-1 cells; 2) Killed bacteria inducer: Dissolve the isolated endophyte RF-1 cells in distilled water to a final concentration of 3 g / L, sterilize at 121°C for 20 min, and cool for later use; 3) Fermentation broth inducer: Concentrate the fermentation broth obtained in S3-1, filter it in a clean bench, and set aside; 4) Yeast powder inducer: Dissolve the endophyte RF-1 powder in distilled water to a final concentration of 3 g / L, filter it in a clean bench, and set aside;
[0018] S3-3: Add any of the elicitors obtained in S3-2 to the adventitious roots cultured in liquid in S2, culture them together with the adventitious roots, collect the adventitious roots, and measure various physiological indicators, biomass, flavonoids, total phenols, and CBD content.
[0019] Among them, preferably, in step S1, the explant disinfection reagent is 75% v / v alcohol and 0.1% v / v mercuric chloride solution; the MS solid culture medium is a solid MS basic culture medium containing 3% w / v sucrose, 2.0 mg / L NAA and 1.0 mg / L IBA; the induction time is 45 days, the induction conditions are 25±3°C, and the light-dark cycle is 16:8.
[0020] Wherein, preferably, in step S2, the liquid MS culture medium is a liquid MS basic culture medium containing 1.0 mg / L NAA and 0.5 mg / L I BA.
[0021] Preferably, in step S3, 1.5×10 7 The endophytes were inoculated into a conical flask containing 100 mL of liquid MS medium. The flask was incubated in the dark on a shaker at 28°C and 130 rpm for 18 days to obtain a fermentation broth of the endophyte RF-1. In step S3-2, the fermentation broth was concentrated 10-fold and filtered in a clean bench for later use to obtain the inducer.
[0022] Among them, preferably, in step S3-3, the physiological indicators include soluble sugar, soluble protein, proline, malondialdehyde, superoxide dismutase and peroxidase, which can be measured according to conventional methods in the art; the flavonoid content is determined by Al(NO3)-NaNO3 colorimetric method, the total phenol content is determined by Folin-phenol method, cannabidiol is qualitatively detected by liquid chromatography-mass spectrometry, and cannabinoid content is detected by high performance liquid chromatography.
[0023] Compared with the prior art, the present invention has the following beneficial effects:
[0024] (1) The present invention can quickly and effectively obtain adventitious roots from industrial hemp explants, and the roots are in good growth condition.
[0025] (2) The present invention screens the most suitable liquid culture system for industrial hemp adventitious roots by establishing a growth curve and orthogonal test, and effectively obtains a stable liquid culture system for industrial hemp adventitious roots.
[0026] (3) The present invention obtains endophytic fungus RF-1 by separation and purification and then induces the adventitious roots of industrial hemp, and obtains significantly increased total CBD, total flavonoids and total phenol contents after induction. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 The growth state of adventitious roots induced by different hormone combinations;
[0028] Figure 2 The root activity of adventitious roots induced under different hormone combinations was investigated, and MS+2.0mg / L NAA+1.0mg / L IBA was determined to be the most suitable medium for root induction.
[0029] Figure 3 The growth curve (A) and growth rate (B) were drawn under MS+1.0mg / LNAA+0.5mg / LIBA, and it was determined that a 30-day period was most suitable for the liquid culture period of industrial hemp adventitious roots.
[0030] Figure 4 The biomass changes after RF-1 induction for 4 days showed that the biomass would be significantly reduced after RF-1 induction for 4 days.
[0031] Figure 5 For the qualitative determination of total CBD content using liquid chromatography-mass spectrometry;
[0032] Among them, A is the standard and B is the sample;
[0033] Figure 6 The total flavonoids (A), total phenols (B) and total CBD (C) contents after RF-1 induction for 4 days;
[0034] Figure 7 This is a heat map of physiological and biochemical indicators after RF-1 induction for 4 days.
[0035] Culture deposit information:
[0036] Strain name: RF-1
[0037] Classification name: Rhodosporidiobolusfluvialis
[0038] Depository: General Microbiology Center of China Culture Collection Administration
[0039] Deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing Deposit number: CGMCCNO.28625
[0040] Storage time: October 13, 2023 DETAILED DESCRIPTION
[0041] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, the embodiments are merely exemplary and do not limit the scope of the present invention in any way. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements fall within the scope of protection of the present invention.
[0042] Example 1: Extraction and identification of endophytes
[0043] 1. Extraction of endophytes:
[0044] S1: Select 2g of upper leaves of industrial hemp in the flowering stage and rinse with tap water for 30min for later use;
[0045] S2: Transfer the industrial hemp leaves prepared in S1 to the clean bench, rinse with sterile water 3-5 times, and use sterile paper to absorb any residual moisture on the surface for later use;
[0046] S3: Disinfect the industrial hemp leaves prepared in S2 with 75% alcohol diluted with sterile water for 30 seconds, remove them, rinse them once with sterile water, and dry the surface moisture with sterile paper for later use;
[0047] S4: Disinfect the industrial hemp leaves prepared in S3 with 0.1% mercuric chloride solution for 5 minutes, then rinse with sterile water 5 times to remove the residual mercuric chloride solution, and dry the residual moisture on the surface with sterile paper for later use;
[0048] S5: Cut the industrial hemp leaves prepared in S4 into pieces with a width of about 0.2 mm using scissors and transfer them into a triangular flask for later use;
[0049] S6: Add 30 ml of a mixture of 1.0% cleavage enzyme and 0.8% mannitol to the S5 flask, seal with parafilm and set aside;
[0050] S7: Place the flask in S6 in a constant temperature shaker at 26°C, 130 rpm / min, and protect from light;
[0051] S8: After 7 hours of enzymatic hydrolysis, the flask was allowed to stand for 5 minutes, and 500 μl of the culture medium was aspirated from the upper layer and inoculated into MS medium (MS basic medium + 3% w / v sucrose) for primary culture. The culture was carried out in a constant temperature shaking incubator at 26°C and 130 rpm / min in the dark.
[0052] 2. Purification and identification of endophytes:
[0053] S1: Select a bacterial solution from MS liquid medium and sterilize the inoculating needle by calcining it in a clean bench. After cooling, dip a small amount of the bacterial solution into the inoculating needle and streak a line on the MS solid medium. After the inoculating needle is calcined and cooled, streak the line again over the first streak. Repeat the above steps once. Do not dip the inoculating needle into the bacterial solution for the next two streaks, only touching the last streak once. Seal the plate with parafilm and incubate in a constant temperature incubator at 27°C in the dark.
[0054] S2: Take a single colony grown in S1 medium, pick up a single colony with an inoculation needle and streak it onto new MS medium for purification. Repeat the purification 3 to 4 times.
[0055] S3: Prepare the culture medium by adding 30 mL of MS liquid culture medium into a 150 mL Erlenmeyer flask and sterilize it by autoclaving at 121°C for 20 min.
[0056] S4: Use a pipette tip to pick up a single colony from the purified S2 and insert it into MS liquid culture medium, shake it, and remove it. After sealing, place it in a constant temperature shaking incubator with continuous shaking at 130 rpm and 27°C in the dark. Culture it for about one to two weeks before subculturing it again.
[0057] S5: Take the purified and stably cultured bacterial solution from S4, culture with shaking for 6 days, take 1 mL of the sample into a 1.5 mL centrifuge tube, centrifuge at 4000 rpm for 3 min, discard the supernatant, add 1 mL of the bacterial solution again and centrifuge to collect the precipitate, repeat the above steps 2 to 3 times, add 1 mL of sterile water to the obtained bacterial precipitate to resuspend the bacteria, centrifuge at 4000 rpm for 3 min, discard the supernatant, add 1 mL of sterile water again to resuspend, centrifuge, and discard the supernatant. The obtained precipitate is frozen in liquid nitrogen for 10 min, and DNA is extracted using a Fungal Genomic DNA Rapid Extraction Kit (Beijing Coolbo Technology Co., Ltd.);
[0058] S7: PCR amplification was performed by selecting fungal universal primers ITS1 and ITS4, fungal 28S rRNA (LSU), and 18S rRNA (SSU) in a 50 μL reaction system;
[0059] S8: The product obtained in S7 was subjected to 0.1% agarose gel electrophoresis at 120V to remove the remaining impurities, and the PCR product was purified and verified. The purified product was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequence determination;
[0060] S9: The sequence results of the amplified products of S8 were evaluated. Chromas software was used to check whether there were overlapping peaks in the sequences. The obtained sequence information was entered into NCBI for BLAST sequence alignment analysis. Strains with high similarity and matching degree were selected from the BLAST database. A phylogenetic tree was constructed using the neighbor-joining method using MEGA-11 software. The species relationship of the strain was determined to be a strain of Rhodosporidiobolusfluvialis (CBS 6568) on the same branch with a similarity ≥96. Combined with the results of morphological observation, the strain was identified as Rhodosporidiobolusfluvialis and named RF-1. The strain has been deposited in the General Microbiology Center of the China Culture Collection Administration, with the deposit number CGMCC NO.28625.
[0061] Example 2 Effectively inducing adventitious root formation of industrial hemp from industrial hemp explants
[0062] This embodiment provides a method for effectively inducing adventitious root formation of industrial hemp from industrial hemp explants, comprising the following steps:
[0063] S1: Select the leaves of industrial hemp before flowering, cut them and rinse them under tap water for 30 minutes;
[0064] S2: Transfer the industrial hemp leaves prepared in S1 to the clean bench, rinse them with sterile water three times, and use sterile paper to absorb the remaining water on the leaves for later use;
[0065] S3: Disinfect the industrial hemp leaves prepared in S2 in 75% v / v alcohol mixed with sterile water for 30 seconds, then remove them with sterilized tweezers, rinse them three times in sterile water, and place them on sterile paper for later use.
[0066] S4: Sterilize the industrial hemp leaves prepared in S3 in 0.1% v / v mercuric chloride solution for 4-5 minutes, then rinse with sterile water 5 times to remove the residual mercuric chloride solution, and dry the residual water with sterile paper for later use;
[0067] S5: Use sterilized scissors to cut the industrial hemp leaves prepared in S4 into 0.5 mm × mm square leaves and place them on sterile paper for later use;
[0068] S6: The industrial hemp leaves prepared in S5 were transferred into solid MS medium with different hormones using sterilized tweezers and sealed with parafilm;
[0069] S7: Place the plate in S6 in an incubator and incubate at room temperature under light;
[0070] S8: The growth status of explants and adventitious roots in the plates was recorded every 10 days (Table 1);
[0071] S9: At 45 days, the growth of industrial hemp explants and adventitious roots on the plate was recorded ( Figure 1 ), and the adventitious roots of industrial hemp were taken to determine the root activity ( Figure 2 );
[0072] Table 1 shows the number of days to rooting, number of root tubers, induction rate, callus rate, and browning rate of adventitious roots induced by different hormone combinations.
[0073]
[0074] Example 2: Establishment of an industrial hemp adventitious root liquid culture system
[0075] This embodiment provides a method for establishing a liquid culture system for adventitious roots of industrial hemp, comprising the following steps:
[0076] S1: Remove the industrial hemp explant from the solid culture medium and place the entire explant with its adventitious roots into a 50 mL liquid culture system containing MS, 2.0 mg / L NAA, and 1.0 mg / L IBA. Place each plate into one bottle.
[0077] S2: After culturing the industrial hemp adventitious roots prepared in S1 in liquid for a period of time, newly grown adventitious roots were obtained;
[0078] S3: Take out the adventitious roots obtained in S2, place them on sterile paper to absorb the culture medium, then rinse them with sterile water 2-3 times to clean the remaining culture medium components, and place them on sterile paper for later use;
[0079] S4: The adventitious roots in S3 were divided into 0.5 g portions, placed in MS liquid culture medium with different hormone combinations using sterile tweezers, and sealed with parafilm;
[0080] S5: Place the flask in S4 in a constant temperature shaker at 25°C and 100 rpm / min for dark incubation;
[0081] S6: Record the changes in adventitious root morphology, color, and texture every 10 days in liquid culture (Table 2), and determine that MS + 1.0 mg / L NAA + 0.5 mg / L IBA is the most suitable hormone combination for adventitious root liquid culture;
[0082] S7: Take the adventitious root in S6 and repeat step S3;
[0083] S8: Divide the adventitious roots from S7 into 0.5 g portions and place them into 50 mL of liquid culture medium containing MS, 1.0 mg / L NAA, and 0.5 mg / L IBA using sterile tweezers.
[0084] S9: Starting from the 7th day of liquid culture, the adventitious roots in S8 were collected every 3 days to measure the changes in biomass and calculate the growth rate ( Figure 3 );
[0085] S10: Repeat steps S3, S8, and S5 during subculture to stably culture industrial hemp adventitious roots;
[0086] Table 2 shows the growth status of adventitious roots in liquid culture under different hormone combinations
[0087]
[0088]
[0089] Note: Root Growth: ++++ indicates 5-10 cm of extension, vigorous root growth, adventitious roots emerging from the callus, with lateral branches and clusters. +++ indicates 3-5 cm of extension, good root growth, dense adventitious roots, lateral branches and clusters. ++ indicates 1-2 cm of extension, good root growth, significant extension from the original root base, but not dense, with lateral branches and no clusters. + indicates small but growing roots, not dense, with no lateral branches and no clusters. - indicates no root changes.
[0090] Example 3: Inducing industrial hemp adventitious roots to produce secondary metabolites
[0091] 1. Preparation of inducers and induction methods
[0092] S1: 1.5×10 7 The endophyte RF-1 isolated in Example 1 was inoculated into a 250 mL conical flask containing 100 mL of liquid MS medium, and the conical flask was placed in a shaker at 28°C and 130 rpm for dark culture for 18 days to obtain a fermentation broth of the endophyte RF-1.
[0093] S2: The fermentation broth of endophyte RF-1 obtained in S1 was centrifuged at 12,000 rpm for 10 min using an ultra-high-speed low-temperature centrifuge to separate the endophyte RF-1 cells from the culture medium and collect the endophyte RF-1 cells;
[0094] S3: The endophyte RF-1 bacteria were collected from the fermentation broth of S1 or S2, and the following four inducers were prepared respectively: 1) Live bacteria inducer: 0.75×10 7Endophyte RF-1; 2) Killed bacteria inducer: Dissolve the isolated endophyte RF-1 cells in distilled water to a final concentration of 3 g / L, sterilize at 121°C for 20 min, and cool for later use; 3) Fermentation broth inducer: Concentrate the fermentation broth obtained in S1 10-fold, filter in a clean bench, and set aside; 4) Yeast powder inducer: Dissolve the endophyte RF-1 powder in distilled water to a final concentration of 3 g / L, filter in a clean bench, and set aside;
[0095] S4: Take out the adventitious roots of industrial hemp cultured in liquid in Example 2 in the clean bench, pour out the old culture medium, and place the adventitious roots on sterile paper for later use;
[0096] S5: Rinse the adventitious roots of S4 with sterile distilled water three times to clean the old culture medium components, absorb excess water with sterile paper, and cut them into 0.5g pieces with sterile tweezers. Weigh them on an electronic scale for later use;
[0097] S6: Place the adventitious roots weighed in S4 into 50 mL of MS medium in a newly prepared 150 mL conical flask, seal the flask with parafilm, and culture in a constant temperature shaker at 100 rpm in the dark for 30 days.
[0098] S7: In a clean bench, add different inducers prepared in S3 to the adventitious roots harvested in S6, seal with parafilm, and place the conical flask in a constant temperature shaker at 100 rpm for dark culture for 4 days.
[0099] 2. Extraction of secondary metabolites from industrial hemp adventitious roots:
[0100] S1: The adventitious roots harvested from the induction culture medium were taken out, the excess culture medium was vacuum pumped out, the fresh weight was measured by electronic balance, and the roots were dried in a constant temperature drying oven at 60°C. The dry weight was measured by electronic balance ( Figure 4 );
[0101] S2: Take the dry weight of the adventitious roots collected in S1, grind them into powder in a mortar, weigh them using an electronic balance, and transfer them to a dry EP tube for later use;
[0102] S3: Take the powder in S2 and dissolve 0.2 g of powder in 2 mL of methanol into an EP tube;
[0103] S4: Take the EP tube in S3 and place it in a constant temperature shaker at 100 rpm for 1 hour;
[0104] S5: Place the EP tube from S4 in an ultracentrifuge at 12,000 rpm for 10 minutes. The supernatant was collected and filtered through a 0.22 μM organic filter. CBD was then qualitatively analyzed by liquid chromatography-mass spectrometry (LC-MS). Cannabidiolic acid (CBDA), a precursor of CBD, is formed after decarboxylation. Total CBD content can typically be calculated as CBD + CBDA × 0.877. The LC-MS column used was a Thermo Scientific™ Hypersil GOLD C18 Colμmn (50 × 2.1 mM, 1.9 μM). Gradient elution (30% B:70% A over 3 minutes; 60% B:40% A over 50 minutes, followed by 90% B:10% A for 2 minutes, then returning to the initial conditions) was used. The flow rate was 0.3 mL / min, the injection volume was 10 μL, and the column temperature was 50°C. Mass spectrometry conditions: ion source: electrospray ion source (ESI), positive ion mode acquisition, nebulizer pressure: 3.5 kV, capillary temperature: 320°C, purge gas flow rate: 20, auxiliary gas flow rate: 5.0, parent ion fragmentation energy: 40 eV, secondary quantitative ion pair: positive ion: 315.2 / 193.0 (m / z), select the example acquisition mode, select the final charge-to-mass ratio CBD of the molecular ion peak to be 193.1222. The qualitative determination results of the LC-MS instrument are as follows: Figure 5 shown.
[0105] The total CBD was quantitatively determined by high performance liquid chromatography (HPLC) using a ZORBAX SB-C18 column (4.6×150 mm, 5 μm) for analysis. The mobile phases were A: 0.1% phosphoric acid in acetonitrile and B: 0.1% phosphate aqueous solution, and gradient elution was used. The elution conditions were as follows: 0-15 min, A was 70%-75%; 15-20 min, A was 75%-90%; 20-23 min (90%-100%); 23-25 min (100%). The detection wavelength was set to 220 nm while maintaining a flow rate of 1.0 mL / min. The injection volume used was 10 μL. The CBD standard curve determined was: y=40.362x+26.266(R 2 =0.9999); CBDA standard curve is: y = 50.8669x - 1.0934 (R 2 =1).
[0106] HPLC detection of total CBD content Figure 6 As shown in C;
[0107] S6: Take the powder from S2 and dissolve 1.0 g of powder in 10 mL of 60% ethanol in an EP tube for extraction. Place the tube in a constant temperature shaker at 100 rpm for 1 h.
[0108] S7: Take the EP tube in S6 and place it in an ultrasonic cleaner for 45 minutes;
[0109] S8: Repeat steps S6 and S7 2 times, centrifuge at 12000 rpm for 10 min, and collect the supernatant for later use;
[0110] S9: Take 1 mL of the extract from S8 and place it in a 10 mL volumetric flask. Add 0.3 mL of 5% Na2NO3 (let it stand for 6 min), 0.3 mL of 10% Al(NO)3 (let it stand for 6 min), and 4 mL of 4% NaOH in sequence. Mix well and make up to 10 mL. Let it stand for 10 min. Determine the total phenol content (OD510 nm) using a UV spectrophotometer. Figure 6 B);
[0111] S10: Take 0.5 mL of the extract from S8 and place it in a 10 mL volumetric flask. Add 0.5 mL of Folin-phenol (mix for 30 seconds) and 1.0 mL of 15% Na2CO3 in sequence. Make up to volume with distilled water and incubate at 20°C in the dark for 1 hour. Determine the total flavonoid content (OD760 nm) using a UV spectrophotometer. Figure 6 A).
[0112] S11: Physiological indicators refer to soluble sugar (anthrone colorimetric method), soluble protein (Coomassie brilliant blue method), proline (Pro), malondialdehyde (MDA), superoxide dismutase (SOD), and peroxidase (POD). The determination methods refer to "Principles and Techniques of Plant Physiology and Biochemistry Experiments". The results are as follows: Figure 7 shown.
[0113] In summary, the content of metabolites in the adventitious roots of industrial hemp increased significantly after induction with the fermentation broth of endophyte RF-1.
Claims
1. An endophyte isolated from industrial hemp explants, named RF-1, was classified as Rhodosporidiobolus fluvialis and deposited in the General Microbiology Center of the China Culture Collection Administration with the deposit number CGMCC NO. 28625.
2. Use of the endophyte according to claim 1 in inducing industrial hemp adventitious roots to produce flavonoids and phenolic substances.
3. The use according to claim 2, characterized in that The phenolic substances include cannabidiol (CBD).
4. A method for inducing industrial hemp adventitious roots to produce flavonoids and phenolic substances, characterized in that: The following steps are involved: S1: Induction of adventitious roots of industrial hemp The above-ground leaves of industrial hemp before flowering were selected as explants, washed and disinfected, and then inoculated into MS solid medium containing NAA and IBA to induce adventitious roots. S2: Liquid culture of adventitious roots Select adventitious roots with good growth status and inoculate them into liquid MS medium for liquid culture. When the adventitious roots grow to fill the bottle, remove the explant part on the adventitious roots and place them into new liquid MS medium for subculture. S3: Preparation and induction of inducers S3-1: inoculating the endophyte of claim 1 into a liquid MS culture medium and culturing the culture to obtain a fermentation broth of the endophyte RF-1; centrifuging the obtained fermentation broth of the endophyte RF-1 using an ultra-high-speed low-temperature centrifuge to separate the endophyte RF-1 cells from the culture medium, and collecting the endophyte RF-1 cells; S3-2: The fermentation broth obtained in S3-1 and the collected endophyte RF-1 bacteria were used to prepare the following four inducers: 1) Live bacteria inducer: 0.75×10 7 1) endophyte RF-1 cells; 2) Killed bacteria inducer: Dissolve the isolated endophyte RF-1 cells in distilled water to a final concentration of 3 g / L, sterilize at 121°C for 20 min, and cool for later use; 3) Fermentation broth inducer: Concentrate the fermentation broth obtained in S3-1, filter it in a clean bench, and set aside; 4) Yeast powder inducer: Dissolve the endophyte RF-1 powder in distilled water to a final concentration of 3 g / L, filter it in a clean bench, and set aside; S3-3: Add any of the elicitors obtained in S3-2 to the adventitious roots cultured in liquid in S2, culture them together with the adventitious roots, collect the adventitious roots, and measure various physiological indicators, biomass, flavonoids, total phenols, and CBD content.
5. The method according to claim 4, wherein In step S1, the explant disinfection reagent is 75% v / v alcohol and 0.1% v / v mercuric chloride solution; the MS solid culture medium is a solid MS basic culture medium containing 3% w / v sucrose, 2.0 mg / L NAA and 1.0 mg / L IBA; the induction time is 45 days, the induction conditions are 25±3°C, and the light-dark cycle is 16:
8.
6. The method according to claim 4, wherein In step S2, the liquid MS culture medium is a liquid MS basic culture medium containing 1.0 mg / L NAA and 0.5 mg / L I BA.
7. The method according to claim 4, wherein In step S3, 1.5×10 7 The endophyte of claim 1 is inoculated into a conical flask added with 100 mL of liquid MS medium, and the conical flask is placed in a shaker at 28° C. and 130 rpm for dark culture for 18 days to obtain a fermentation broth of the endophyte RF-1; in step S3-2, the fermentation broth is concentrated 10 times and filtered in a clean bench for later use to obtain an inducer.
8. The method according to claim 4, wherein In step S3-3, the physiological indicators include soluble sugar, soluble protein, proline, malondialdehyde, superoxide dismutase and peroxidase, which can be measured according to conventional methods in the art; the flavonoid content is determined by Al(NO3)-NaNO3 colorimetric method, the total phenol content is determined by Folin-phenol method, cannabidiol is qualitatively detected by liquid chromatography-mass spectrometry, and cannabinoid content is detected by high performance liquid chromatography.