A method for cultivating adventitious roots of industrial hemp with high yields of cannabinoids and tanshinone

Abstract

This application relates to a method for cultivating high-yield cannabinoid and tanshinone adventitious roots of industrial hemp, belonging to the field of adventitious root cultivation technology. The method includes inducing industrial hemp adventitious roots with yeast extract to obtain induced adventitious roots. Yeast extract is low-cost and readily available, and as an inducer, it can stimulate industrial hemp adventitious roots to synthesize more target metabolites. The induced adventitious roots are then scaled up and cultured in a bioreactor, promoting cell proliferation and growth, and increasing the biomass of the adventitious roots. Simultaneously, it promotes the accumulation of more target metabolites in the adventitious roots, with yields of cannabinoids, cannabidiol, tetrahydrotanshinone I, tanshinone IIA, and dihydrotanshinone I reaching as high as 80.65, 53.58, 103.76, 21.71, and 5.71 mg / L DW, respectively. This method saves on operational steps and labor intensity, and overcomes the shortcomings of low biomass and low cannabinoid yield in existing industrial hemp adventitious root culture technology. It can achieve year-round economical and efficient cultivation of industrial hemp adventitious roots with high yields of cannabinoids and tanshinone.

Description

Technical Field

[0001] This application relates to the field of adventitious root cultivation, and more particularly to a method for cultivating industrial hemp adventitious roots that produce high yields of cannabinoids and tanshinone. Background Technology

[0002] Industrial hemp (Cannabas satava L.), an annual herbaceous plant, contains abundant bioactive substances, including cannabinoids, flavonoids, phenols, alkaloids, and terpenes, and has been widely used in food, pharmaceutical research and development, and cosmetics. Cannabinoids are representative compounds in industrial hemp. Currently, to replace traditional industrial hemp cultivation methods, existing technologies show that adventitious roots of industrial hemp cultivated in bioreactors can produce cannabinoids. However, these technologies still suffer from low cannabinoid content and low adventitious root biomass, which limits the commercial application of industrial hemp adventitious roots for cannabinoid production. Therefore, it is urgent to overcome the shortcomings of existing adventitious root cultivation technologies. Summary of the Invention

[0003] This application provides a method for cultivating industrial hemp adventitious roots with high yields of cannabinoids and tanshinone to solve the following technical problem: how to simultaneously increase the biomass of industrial hemp adventitious roots and the yields of cannabinoids and tanshinone.

[0004] This invention provides a method for cultivating adventitious roots of industrial hemp that produce high yields of cannabinoids and tanshinone, comprising the following steps:

[0005] Yeast extract was used to induce the adventitious roots of industrial hemp, resulting in induced adventitious roots of industrial hemp.

[0006] A bioreactor was used to scale up the cultivation of adventitious roots of induced industrial hemp.

[0007] Collect adventitious roots of industrial hemp after large-scale cultivation.

[0008] Optionally, the step of inducing industrial hemp adventitious roots with yeast extract to obtain induced industrial hemp adventitious roots specifically includes:

[0009] Under aseptic conditions, adventitious roots of industrial hemp were inoculated into shake flasks containing liquid culture medium and cultured to obtain adventitious roots of industrial hemp in suspension.

[0010] Yeast extract was added to the suspended cultured adventitious roots of industrial hemp and cultured for another 48 to 96 hours to obtain the induced industrial hemp adventitious roots.

[0011] Optionally, the amount of yeast extract added is 300 mg to 400 mg per liter of liquid culture medium.

[0012] Optionally, the process of using suspension culture technology to cultivate adventitious roots of industrial hemp to obtain an industrial hemp adventitious root suspension culture system specifically includes: placing industrial hemp adventitious roots in a shake flask containing liquid culture medium and shaking and incubating in the dark for 30 to 40 days to obtain an industrial hemp adventitious root suspension culture system.

[0013] Optionally, the temperature for shaking and dark incubation is 20℃~30℃, and the rotation speed is 90r / min~110r / min.

[0014] Optionally, the process of using a bioreactor to scale up the adventitious roots of induced industrial hemp specifically includes:

[0015] The induced adventitious roots of industrial hemp were inoculated into liquid culture medium in a bioreactor for the first scale-up culture.

[0016] The adventitious roots of industrial hemp after the first amplification culture were used as experimental materials, inoculated into a bioreactor, and a second amplification culture was carried out using a fed-batch culture mode.

[0017] Optionally, the inoculation density of the industrial hemp adventitious roots is 10 g / L to 15 g / L.

[0018] Optionally, the first scale-up culture shall meet at least one of the following conditions: the culture temperature is 20℃~30℃, the culture time is 40d~50d, and the aeration rate is 0.06vvm~0.08vvm.

[0019] Optionally, the second scale-up culture using a fed-batch mode specifically includes: after an initial culture period, adding feed medium to the bioreactor, and then carrying out fed-batch culture;

[0020] The second amplification culture must meet at least one of the following conditions: the culture temperature is 20℃~30℃, the aeration rate is 0.06vvm~0.08vvm, the initial culture time is 45d~55d, the amount of feed medium added is 25%~35% of the initial culture medium, and the feed culture time is 30d~40d.

[0021] Optionally, the liquid culture medium and the feed culture medium comprise: 0.75×MS–MS medium, 2 mg / L to 3 mg / L IBA and 25 g / L to 35 g / L sucrose, with a pH of 5 to 6.

[0022] The technical solution provided in this application has the following advantages compared with the prior art:

[0023] This application relates to a method for cultivating high-yield cannabinoid and tanshinone adventitious roots of industrial hemp, belonging to the field of adventitious root cultivation technology. The method includes inducing industrial hemp adventitious roots with yeast extract to obtain induced adventitious roots. Yeast extract is low-cost and readily available, and as an inducer, it can stimulate industrial hemp adventitious roots to synthesize more target metabolites. The induced adventitious roots are then scaled up and cultured in a bioreactor, promoting cell proliferation and growth, and increasing the biomass of the adventitious roots. Simultaneously, it promotes the accumulation of more target metabolites in the adventitious roots, with yields of cannabinoids, cannabidiol, tetrahydrotanshinone I, tanshinone IIA, and dihydrotanshinone I reaching as high as 80.65, 53.58, 103.76, 21.71, and 5.71 mg / LDW, respectively. This method saves on operational steps and labor intensity, and overcomes the shortcomings of low biomass and low cannabinoid yield in existing industrial hemp adventitious root culture technology. It can achieve year-round economical and efficient cultivation of industrial hemp adventitious roots with high yields of cannabinoids and tanshinone. Attached Figure Description

[0024] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0025] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a schematic flowchart of a method for cultivating industrial hemp adventitious roots with high yields of cannabinoids and tanshinone, according to some embodiments of this application.

[0027] Figure 2 These are the standard precursor ion mass spectra and MRM chromatograms from the UPLC-QqQ-MS / MS detection examples, including cannabinoid compounds (CBG, CBD, and CBN) and tanshinone compounds (tanshinone IIA, tetrahydrotanshinone I, and dihydrotanshinone I).

[0028] Figure 3 This is a schematic diagram illustrating the effects of yeast extract (YE) treatment concentration and treatment time on the adventitious root growth (a) and the accumulation of cannabinoids and tanshinone (b) of industrial hemp in the examples. Data represent mean ± standard deviation (n=3). Significant differences exist in mean ± standard deviation for data that do not share the same lowercase letters (p<0.05).

[0029] Figure 4This is a schematic diagram illustrating the effects of yeast extract (YE) on the content (a) and yield (b) of cannabinoids and tanshinone in adventitious roots of industrial hemp cultured in a bioreactor, as well as the observation of the morphological structure of adventitious roots (c).

[0030] Figure 5 This diagram illustrates the effect of initial culture time on the adventitious root biomass (a and b) of industrial hemp cultured in a bioreactor, as well as its cannabinoid and tanshinone content (c) and yield (d). Data are presented as mean ± standard deviation (n = 3). Significant differences exist in mean ± standard deviation for data not sharing the same lowercase letters (p < 0.05).

[0031] Figure 6 This is a schematic diagram illustrating the effect of feed medium volume on the adventitious root biomass (a and b) of industrial hemp cultured in a bioreactor, as well as its cannabinoid and tanshinone content (c) and yield (d). Data are presented as mean ± standard deviation (n = 3). Significant differences exist in mean ± standard deviation for data not sharing the same lowercase letters (p < 0.05).

[0032] Figure 7 This is a schematic diagram illustrating the effect of fed-culture time on the adventitious root biomass (a and b) of industrial hemp cultured in a bioreactor, as well as its cannabinoid and tanshinone content (c) and yield (d). Data are presented as mean ± standard deviation (n = 3). Significant differences exist in mean ± standard deviation for data that do not share the same lowercase letters (p < 0.05).

[0033] Figure 8 This is a schematic diagram illustrating the effects of untreated and treated yeast extracts, as well as fed-batch culture, on the adventitious root growth of industrial hemp in the embodiments.

[0034] Figure 9 This is a schematic diagram illustrating the effects of continuous culture mode with and without yeast extract on the yield of cannabinoids and tanshinone in adventitious roots of industrial hemp in the examples, comparing the effects of continuous culture mode with and without yeast extract on the yield of cannabinoids and tanshinone in industrial hemp. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0036] Various embodiments of this application may exist in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as a hard limitation on the scope of this application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within that range; for example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single numbers within the range such as 1, 2, 3, 4, 5, and 6, regardless of the range; in addition, whenever a numerical range is indicated herein, it means including any referenced number (fraction or integer) within the indicated range.

[0037] In this document, terms such as “comprising” mean “including but not limited to”. Relational terms such as “first” and “second” are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. “And / or” describes the relationship between related objects, indicating that there can be three relationships, for example, A and / or B can mean: A alone, A and B simultaneously, or B alone; where A and B can be singular or plural. “At least one” means one or more, “more” means two or more; “at least one,” “at least one of the following,” or similar expressions refer to any combination of these items, including any combination of single or plural items; for example, “at least one of a, b, or c,” or “at least one of a, b, and c,” can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple. "Parts representation" such as parts by weight or parts by mass indicates the proportional relationship between components. In the proportional relationships discussed in this article, the parameters that need to be described by proportion should be understood as the first term of the proportion in the order of description, and the proportion figures should be understood as the second term of the proportion. For example, if the mass ratio of substance A, substance B, and substance C is 1:2:3, then substances A, B, and C should correspond one-to-one with the proportion figures in the proportion in the order of description, that is, the mass of substance A: the mass of substance B: the mass of substance C = 1:2:3.

[0038] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this article can be purchased from the market or prepared by existing methods.

[0039] This invention provides a method for cultivating adventitious roots of industrial hemp that produce high yields of cannabinoids and tanshinone, comprising the following steps:

[0040] Yeast extract was used to induce the adventitious roots of industrial hemp, resulting in induced adventitious roots of industrial hemp.

[0041] A bioreactor was used to scale up the cultivation of adventitious roots of induced industrial hemp.

[0042] Collect adventitious roots of industrial hemp after large-scale cultivation.

[0043] In the above embodiments, yeast extract is used as an inducer because it is low in cost and readily available. As an inducer, it can stimulate the adventitious roots of industrial hemp to synthesize more target metabolites. The inducing treatment of the industrial hemp adventitious roots is then scaled up using a bioreactor, promoting cell proliferation and growth, and increasing the biomass of the adventitious roots. Simultaneously, it promotes the accumulation of more target metabolites in the adventitious roots. At this point, the yields of cannabidiol, cannabidiol, tetrahydrotanshinone I, tanshinone IIA, and dihydrotanshinone I in the adventitious roots reach as high as 80.65, 53.58, 103.76, 21.71, and 5.71 mg / L DW, respectively. This method saves on operational steps and labor intensity, overcoming the shortcomings of low biomass and low cannabinoid yield in existing industrial hemp adventitious root culture technologies. It enables the economical and efficient cultivation of high-yield cannabinoid and tanshinone industrial hemp adventitious roots year-round.

[0044] As an optional implementation, the step of inducing industrial hemp adventitious roots with yeast extract to obtain induced industrial hemp adventitious roots specifically includes:

[0045] Under aseptic conditions, adventitious roots of industrial hemp were inoculated into shake flasks containing liquid culture medium and cultured to obtain adventitious roots of industrial hemp in suspension.

[0046] Yeast extract was added to the suspended cultured adventitious roots of industrial hemp and cultured for another 48 to 96 hours to obtain the induced industrial hemp adventitious roots.

[0047] As an optional implementation, the amount of yeast extract added is 300 mg to 400 mg of yeast extract per liter of liquid culture medium.

[0048] In the above embodiments, the reason for controlling the amount of yeast extract added is that when the amount of yeast extract added is 300 mg to 400 mg per liter of liquid culture medium, the adventitious roots greatly promote the accumulation of cannabinoids and tanshinone. When the amount of yeast extract added is less than 300 mg / L, the concentration of yeast extract in the culture medium is low and cannot fully promote the synthesis of target metabolites by adventitious roots. When the amount of yeast extract added is higher than 400 mg / L, the concentration of yeast extract in the culture medium is high, and high concentrations of yeast extract contain a large amount of nutrients such as amino acids, vitamins, and minerals, which may lead to nutrient overload; at the same time, some components in yeast extract may inhibit the production of target metabolites by adventitious roots.

[0049] For example, the amount of yeast extract added per liter of liquid culture medium can be set to 300 mg, 310 mg, 32 mg, 330 mg, 340 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg or 400 mg.

[0050] As an optional implementation, the method of culturing industrial hemp adventitious roots using suspension culture technology to obtain an industrial hemp adventitious root suspension culture system specifically includes: placing 20g / L–40g / L of industrial hemp adventitious roots into a shake flask containing liquid culture medium, and shaking and incubating in the dark for 30d–40d to obtain an industrial hemp adventitious root suspension culture system.

[0051] In the above embodiments, the adventitious roots of industrial hemp are first subjected to suspension culture. Since the number of adventitious roots is small, the inoculation density is 20g / L–40g / L. For example, the inoculation density of industrial hemp adventitious roots during suspension culture can be set to 20g / L, 22g / L, 24g / L, 25g / L, 26g / L, 28g / L, 30g / L, 32g / L, 34g / L, 35g / L, 36g / L, 38g / L, or 40g / L.

[0052] As an optional implementation, the temperature for shaking dark incubation is 20℃~30℃, and the rotation speed is 90r / min~110r / min.

[0053] In the above embodiments, the temperature for shaking and dark culture of industrial hemp adventitious roots can be set to 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃ or 30℃, preferably 25±2℃; the rotation speed can be set to 90r / min, 95r / min, 100r / min, 105r / min or 110r / min.

[0054] As an optional implementation, the scale-up cultivation of induced industrial hemp adventitious roots using a bioreactor specifically includes:

[0055] The induced adventitious roots of industrial hemp were inoculated into liquid culture medium in a bioreactor for the first scale-up culture.

[0056] The adventitious roots of industrial hemp after the first amplification culture were used as experimental materials, inoculated into a bioreactor, and a second amplification culture was carried out using a fed-batch culture mode.

[0057] In the above embodiments, during the first scale-up culture, the adventitious roots of industrial hemp after induction treatment were inoculated into a bioreactor containing liquid culture medium. After the adventitious roots were cultured in a light-protected environment for 40 to 50 days, the biomass of the adventitious roots and the content of their target metabolites increased significantly, and the liquid culture medium was absorbed and depleted by the adventitious roots.

[0058] During the second scale-up culture, a fed-batch culture mode was adopted. A certain amount of adventitious roots from the first scale-up culture were inoculated into the bioreactor and the second scale-up culture was continued under dark conditions.

[0059] Adding the culture medium to the bioreactor in batches can provide a continuous and stable environment for the growth of adventitious roots.

[0060] As an optional implementation, during scale-up cultivation, the inoculation density of the adventitious roots of the industrial hemp is 10 g / L to 15 g / L.

[0061] In the above embodiments, the reason for controlling the inoculation density of industrial hemp adventitious roots is that when the inoculation density is greater than 15 g / L, the nutrient supply of the liquid culture medium is insufficient, affecting the growth of adventitious roots. When the inoculation density is less than 10 g / L, it results in waste of liquid culture medium. For example, the inoculation density of industrial hemp adventitious roots can be set to 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, or 15 g / L.

[0062] As an optional implementation, the first scale-up culture shall at least meet one of the following conditions: the culture temperature is 20℃~30℃, the culture time is 40d~50d, and the aeration rate is 0.06vvm~0.08vvm.

[0063] In the above embodiments, the temperature for the first scale-up culture can be set to 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃ or 30℃, preferably 25±2℃; the culture time can be adjusted according to the growth of adventitious roots, for example, the culture time can be 40d, 41d, 42d, 43d, 44d, 45d, 46d, 47d, 48d, 49d or 50d; the aeration rate can be set to 0.06vvm, 0.07vvm or 0.08vvm.

[0064] As an optional implementation, the second scale-up culture using a fed-batch mode specifically includes: after an initial culture period, adding feed medium to the bioreactor, and then carrying out fed-batch culture;

[0065] The second amplification culture must meet at least one of the following conditions: the culture temperature is 20℃~30℃, the aeration rate is 0.06vvm~0.08vvm, the initial culture time is 45d~55d, the amount of feed medium added is 25%~35% of the volume of the initial culture medium, and the feed culture time is 30d~40d.

[0066] In the above embodiments, the temperature and aeration rate of the second amplification culture are the same as those of the first amplification culture. The main difference is that the initial culture is carried out for 45 to 55 days. This time period can be set according to the actual consumption of culture medium, such as 45 days, 48 ​​days, 50 days, 53 days or 55 days for the initial culture. The amount of supplemental culture medium added is 25%–35% of the initial culture medium volume. When the amount of supplemental culture medium added is less than 25%, the biomass of adventitious roots increases with the increase of the amount added, reaching its maximum at 25%, but at this point, most of the target metabolites accumulated are not in the optimal state. When the amount of supplemental culture medium added is greater than 35%, neither the biomass of adventitious roots nor the target metabolites are significantly increased. Therefore, the supplemental culture medium can be set to 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% of the initial culture medium volume. The supplemental culture time can be set to 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days according to the actual situation.

[0067] As an optional implementation, the liquid culture medium and the feed culture medium can be conventional culture media, or may include: 0.75×MS–MS medium, 2 mg / L to 3 mg / L IBA and 25 g / L to 35 g / L sucrose, with a pH of 5 to 6.

[0068] The present application is further illustrated below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national / industry standards; if there is no corresponding national / industry standard, they are performed according to general international standards, conventional conditions, or conditions recommended by the manufacturer.

[0069] Example 1

[0070] This embodiment provides a method for cultivating adventitious roots of industrial hemp that produce high yields of cannabinoids and tanshinone, including the following steps:

[0071] Take 20 g / L of industrial hemp adventitious roots and place them in a shake flask containing liquid culture medium. Incubate in the dark at 25℃ and 110 r / min for 40 days.

[0072] The liquid culture medium included 3 / 4MS medium, 2 mg / L IBA and 25 g / L sucrose, with a pH of 5.

[0073] Add 300 mg / L of yeast extract to the liquid culture medium in the shake flask from the previous step, and continue to suspend and culture the adventitious roots for 96 h. Use yeast extract to induce the adventitious roots of industrial hemp to obtain the induced adventitious roots of industrial hemp.

[0074] Under aseptic conditions, adventitious roots of industrial hemp after induction treatment were inoculated into a bioreactor for the first scale-up culture. The inoculation density was 10 g / L, the culture time was 40 days, and the adventitious roots were collected.

[0075] The adventitious roots of industrial hemp after the first scale-up culture were used as the experimental material for the second scale-up culture. 10 g / L adventitious roots were inoculated into the bioreactor. After the initial culture of 45 days, the feed medium was added to the bioreactor and then fed culture was carried out for 30 days.

[0076] Collect the adventitious roots of industrial hemp after the second amplification culture.

[0077] The contents of cannabinoids (CBD, CBG, CBN) and tanshinones (tanshinone IIA, dihydrotanshinone I, and tetrahydrotanshinone I) in the adventitious roots of industrial hemp were determined using UPLC-QqQ-MS / MS. The specific testing method is shown below:

[0078] Sample preparation:

[0079] The standards were accurately weighed and dissolved in UPLC-MS grade methanol to prepare solutions, which were then serially diluted to obtain standard solutions of the corresponding concentrations. Adventitious root materials from the experimental group, after being lyophilized, were ground, and 0.1 g was weighed and mixed with 3 mL of methanol-dichloromethane (9:1, v / v). Extraction was then performed by sonication at 0 °C for 60 min, followed by centrifugation at 12000 rpm for 8 min. The supernatant was then freeze-dried in 0.5 mL of UPLC-MS grade methanol for 12 h and centrifuged (12000 rpm, 8 min) to obtain the extraction solution. Subsequently, the standard and sample solutions were filtered through a 0.22 μm PVDF membrane and transferred to automated sampler vials for UPLC-MS / MS analysis.

[0080] Instruments and analytical methods

[0081] Analysis was performed using an Agilent 1290 ultra-high performance liquid chromatograph and an Agilent 6460 triple quadrupole mass spectrometer (Agilent Technologies, USA) with an autosampler. The ion source was set to electrospray ionization (ESI), and all analytes were operated in either positive or negative ion mode. Agilent Mass Hunter workstation software (version B.07.00) was used as the data system. Chromatographic separations were performed using an Agilent ZORBAX Eclipse Plus C18 column (50 mm, 2.1 mm ID, 1.8 μm) at 30 °C. The mobile phase flowed at a rate of 0.4 mL / min and consisted of a linear gradient of 0.1% (v / v) formic acid aqueous solution (a) and acetonitrile (B): 0–1.0 min, 5% B v / v; 1.0–5.0 min, 5–95% B v / v; 5.0–6.0 min, 95% B v / v; 6.0–7.0 min, 95–5% B (v / v). The column was flushed for 3 min before each injection. Optimized ESI conditions were set as follows: gas temperature 350 °C, gas flow rate 10 L / min, nebulizer pressure 50 psi, and battery acceleration voltage 4 V. Specific MRM parameters for cannabinoid and tanshinone standards were optimized using Agilent software, including the selection of precursor / product ion combinations, fragment voltage, and collision energy (Table 1 and...). Figure 2 ).

[0082] Table 1. Optimized Multiple Reaction Monitoring (MRM) Parameters for Cannabinoid and Tanshinone Compound Standards

[0083]

[0084] Calibration curves were established based on the peak areas of six concentrations of standard solutions containing cannabinoids (CBD, CBG, CBN) and tanshinones (tanshinone IIA, dihydrotanshinone I, and tetrahydrotanshinone I). Unweighted least squares linear regression was used to investigate linearity, and the resulting standard curves were y = 0.126x ± 0.061 (R²). 2 =0.9993), y=4.194x+0.191(R 2 =0.9989), y=3.15x±0.021(R 2 =0.9998), y=0.132x+0.093(R 2 =0.9991), y=0.715x+0.024(R 2 =0.9992) and y = 3.319x + 0.693(R) 2 =0.9996). Using the formula: content × dry weight / volume = production, calculate the production of cannabinoids and tanshinone in adventitious roots.

[0085] Experimental Example 1

[0086] This experimental example compares the effects of different amounts of yeast extract on adventitious roots of industrial hemp, and includes the following steps:

[0087] 25 g / L of industrial hemp adventitious roots were inoculated into 6 groups of shake flasks containing liquid culture medium and cultured in the dark at 110 r / min at 25℃ for 30 days.

[0088] Yeast extracts at concentrations of 0, 100, 200, 300, 400 and 500 mg / L were added to the liquid culture medium in the shake flask from the previous step, and the adventitious roots were cultured in suspension for 72 h to obtain the induced industrial hemp adventitious roots.

[0089] The above process was repeated ten times, and the adventitious roots of the induced industrial hemp were collected and tested.

[0090] The test results are shown in Table 2 and Figure 3 As shown:

[0091] Table 2. Effects of yeast extract treatment concentration on adventitious root growth in shake-flask cultured industrial hemp.

[0092] Treatment concentration (mg / L) Fresh weight / FW (g / L) Dry weight / DW (g / L) 0 11.33±0.05 1.19±0.02 50 11.41±0.07 1.23±0.02 100 11.26±0.03 1.17±0.01 200 11.19±0.02 1.11±0.01 300 11.27±0.04 1.18±0.01 400 11.35±0.03 1.21±0.01

[0093] Note: Values ​​are expressed as mean ± standard deviation (n = 10).

[0094] As shown in Table 2, treatment of industrial hemp adventitious roots with different concentrations of yeast extract did not significantly affect the fresh and dry weight of the adventitious roots. Figure 3It is known that, regarding the accumulation of cannabinoids and tanshinones in adventitious roots, the yield of cannabinoids and tanshinones in adventitious roots increases to the highest level as the concentration of yeast extract treatment increases to 300 mg / L. Among them, the most significant increases are seen in tetrahydrotanshinone I, tanshinone IIA, and CBD, which are increased by 1.8 times, 1.8 times, and 1.5 times, respectively. However, when the concentration of yeast extract treatment exceeds 400 mg / L, the accumulation of the target metabolites actually decreases. Therefore, the suitable treatment concentration of yeast extract in this invention is 300 mg / L to 400 mg / L.

[0095] Experiment Example 2

[0096] This experimental example compares the effects of different yeast extract treatment times on adventitious roots of industrial hemp, and includes the following steps:

[0097] 25 g / L of industrial hemp adventitious roots were inoculated into 6 groups of shake flasks containing liquid culture medium and cultured in the dark at 110 r / min at 25℃ for 30 days.

[0098] Yeast extract at a concentration of 300 mg / L was added to the liquid culture medium in the shake flask from the previous step, and the adventitious roots were cultured in suspension for 0 h, 12 h, 24 h, 48 h, 96 h and 192 h to obtain the industrial hemp adventitious roots after induction treatment.

[0099] Adventitious roots of industrial hemp after induction treatment were collected and tested. The test results are shown in Table 3 and... Figure 3 As shown:

[0100] Table 3. Effects of yeast extract treatment time on adventitious root growth in shake-flask cultured industrial hemp.

[0101] Processing time (h) Fresh weight / FW (g / L) Dry weight / DW (g / L) 0 10.95±0.01 1.09±0.01 12 10.97±0.01 1.10±0.01 24 10.99±0.01 1.11±0.02 48 10.94±0.01 1.09±0.01 96 11.02±0.02 1.12±0.03 192 11.04±0.02 1.10±0.01

[0102] Note: Values ​​are expressed as mean ± standard deviation (n = 10).

[0103] The data in Table 3 show that the biomass of adventitious roots from industrial hemp did not change significantly during treatment with yeast extract; from Figure 3 It can be seen that the yields of cannabinoids and tanshinones in adventitious roots reached their highest levels after 48 h and 96 h of yeast extract treatment. Among them, the yields of three tanshinone compounds increased more significantly after 96 h of treatment, namely tanshinone IIA (1.12 times), tetrahydrotanshinone I (1.33 times), and dihydrotanshinone I (1.48 times).

[0104] Experimental Example 3

[0105] This experimental example compares the growth and accumulation of target metabolites of industrial hemp adventitious roots in a bioreactor before and after yeast extract induction treatment, and includes the following steps:

[0106] 25 g / L of industrial hemp adventitious roots were inoculated into 6 shake flasks containing liquid culture medium and cultured in the dark at 110 r / min at 25 °C for 30 days.

[0107] In three of the shake flasks, yeast extract at a concentration of 300 mg / L was added to induce adventitious roots. After culturing for 96 hours, the induced adventitious roots were collected. In the other three shake flasks, the adventitious roots were not induced, and after culturing for 96 hours, the uninduced adventitious roots were collected.

[0108] Under aseptic conditions, the adventitious roots of industrial hemp collected in the previous step and induced by yeast extract were inoculated into a bioreactor containing 3 / 4 MS medium, 3 mg / L IBA and 35 g / L sucrose liquid medium for the first scale-up culture. At the same time, untreated adventitious roots were inoculated into the bioreactor as a control. The inoculation density was 15 g / L. During the culture, the aeration rate was set to 0.08 vvm. Each group was set with three replicates. After 40 days of dark culture, the adventitious roots were collected for investigation and observation of adventitious root morphology and structure.

[0109] Freshly collected adventitious roots were fixed in 2% (v / v) glutaraldehyde for 12 h, centrifuged, and the supernatant was discarded. The adventitious roots were then washed three times with a buffer solution and dehydrated with ethanol (30%, 50%, 70%, 90%, and 100%). The adventitious roots were centrifuged (4℃, 12000 r / min, 5 min) and freeze-dried. An ion sputtering technique was used to deposit a conductive film on the surface of the adventitious roots for 2 min. The morphology and structure of the adventitious roots were observed using a scanning electron microscope. The results are as follows: Figure 4 As shown in (c). Observation Figure 4 (c) The morphological structure of the adventitious roots shows that, compared with the control group, the yeast extract reduced the number of cortical cells in the adventitious roots cultured in the bioreactor, resulting in a smaller central column diameter and altering the structure of the adventitious roots.

[0110] The biomass of adventitious roots was investigated, and the data obtained are shown in Table 4. The data in Table 4 show that the biomass of adventitious roots in the first scale-up culture after induction treatment was significantly higher than that of the control. Yeast extract treatment promoted rapid growth of adventitious roots in the bioreactor culture; however, due to excessive consumption of culture medium during growth, the growth of adventitious roots was restricted, leading to aging. Consequently, the adventitious roots turned brownish-yellow, and the differentiated lateral roots were short and hard. Calculations of the target metabolites were performed, and the results are as follows: Figure 4 (a) and Figure 4As shown in (b), the figure shows that the yields of cannabinoids and tanshinone in the induced adventitious roots were significantly increased compared to the control group.

[0111] Table 4. Effects of yeast extract on adventitious root growth of industrial hemp cultured in a bioreactor.

[0112]

[0113] Note: Values ​​are expressed as mean ± standard deviation (n=3).

[0114] Experiment Example 4

[0115] This experimental example compares the effects of different initial culture times on adventitious roots of industrial hemp in a fed-batch culture model, and includes the following steps:

[0116] Under aseptic conditions, adventitious roots of industrial hemp after the first amplification culture induction treatment (10 g / L) were inoculated into four bioreactors containing 2 L of liquid culture medium. After initial culture for 30, 40, 50 and 60 days, 25% of the initial culture medium volume was added as feed medium. After culturing for another 20 days, the adventitious roots of industrial hemp were collected.

[0117] The liquid culture medium and the fed culture medium have the same composition, including 3 / 4MS medium, 2 mg / L IBA and 25 g / L sucrose, with a pH of 5.

[0118] The biomass and target metabolite content of the collected adventitious roots of industrial hemp were investigated, and the results are as follows: Figure 5 As shown, with the initial culture time increasing from 30 days to 50 days, the fresh weight (124.51±0.5 g / L) and dry weight (12.84±0.2 g / L) of industrial hemp adventitious roots increased to their maximum values. The yields of cannabinoids and tanshinone also showed a similar trend. Compared with the adventitious roots collected in the previous group, the increases in CBD (1.42 times) and tetrahydrotanshinone I (1.63 times) were particularly significant. When the initial culture time was extended to 60 days, the biomass of adventitious roots gradually decreased, and the accumulation of cannabinoids and tanshinone also began to decrease. Therefore, an initial culture time of 45-55 days is suitable.

[0119] Experimental Example 5

[0120] This experimental example compares the effects of different feed medium volumes on adventitious roots of industrial hemp in a fed-batch culture mode, and includes the following steps:

[0121] Under aseptic conditions, adventitious roots of industrial hemp after the first amplification culture induction treatment (10 g / L) were inoculated into four groups of bioreactors containing 2 L of liquid culture medium. After initial culture for 50 days, 10%, 20%, 30%, and 40% of the initial culture medium volume were added to each group of bioreactors as feed medium, with each group set as three replicates. After 20 days of feed culture under the same conditions, the adventitious roots were collected.

[0122] The liquid culture medium and the fed culture medium have the same composition, including 3 / 4MS medium, 2 mg / L IBA and 25 g / L sucrose, with a pH of 5.

[0123] The biomass and metabolite content of the collected adventitious roots were measured, and the results are as follows: Figure 6 As shown, when the volume of the fed culture medium added to the bioreactor increased from 10% to 20% of the initial culture medium volume, the fresh weight and dry weight of adventitious roots increased to their maximum values, reaching 136.51±0.02 g / L and 13.57±0.03 g / L, respectively. When the volume of the fed culture medium increased from 20% to 30%, the biomass of adventitious roots did not change significantly. An investigation into the accumulation of cannabinoids and tanshinone in adventitious roots revealed that the yields of CBD and tanshinone reached their highest when the volume of the fed culture medium was 30% of the initial culture medium volume. The tanshinone yield showed a significant increase compared to the adventitious roots under the previous optimal conditions, with increases of 1.27 times for tetrahydrotanshinone I, 1.24 times for tanshinone IIA, and 1.12 times for dihydrotanshinone I. Only the CBG yield reached its highest value at 20% of the initial culture medium volume, at 43.57±0.18 mg / L DW. Taking all factors into consideration, when cultivating industrial hemp adventitious roots with high yields of cannabinoids and tanshinone on a large scale, an initial culture medium volume of 25% to 35% can be selected as the appropriate supplementary culture medium volume.

[0124] Experimental Example 6

[0125] This experimental example compares the effects of different fed-batch culture times on adventitious roots of industrial hemp in a fed-batch culture model, and includes the following steps:

[0126] Under sterile conditions, after the first amplification culture of the induced industrial hemp adventitious roots, 10 g / L of the collected adventitious roots were inoculated into 4 groups of bioreactors. After the initial culture of 50 days, 30% of the initial culture medium volume was added to each group of bioreactors as feed medium. After culturing for 10 days, 20 days, 30 days and 40 days under the same conditions, three replicates were set up for each group, and the adventitious roots were collected.

[0127] The liquid culture medium and the fed culture medium have the same composition, including 3 / 4MS medium, 2 mg / L IBA and 25 g / L sucrose, with a pH of 5.

[0128] The biomass and metabolite content of the collected adventitious roots were measured, and the results are as follows: Figure 7 As shown, when the fed-batch culture time was increased to 40 days, the accumulation of cannabinoids and tanshinone in adventitious roots was measured. It was found that the content and yield of cannabinoids and tanshinone reached their highest levels at a fed-batch culture time of 30 days. Specifically, the yields of CBG, CBD, and tanshinone IIA increased by 1.85 times, 1.85 times, and 1.47 times respectively compared to adventitious roots under the previous optimal conditions, followed by dihydrotanshinone I (1.31 times). Therefore, the suitable fed-batch culture time is 30 to 40 days.

[0129] Experimental Example 7

[0130] This experimental example compares the effects of fed-batch culture and conventional large-scale culture on adventitious root growth, and includes the following steps:

[0131] Under aseptic conditions, after the first amplification culture of the induced industrial hemp adventitious roots, 10 g / L of the collected industrial hemp adventitious roots from the first amplification culture were inoculated into three groups of bioreactors containing 2 L of liquid culture medium. After the initial culture in one group of bioreactors was carried out for 50 days, 30% of the initial culture medium volume was added as feed medium. After another 30 days of culture, the adventitious roots were collected as industrial hemp adventitious roots for the second amplification culture.

[0132] The other two groups were treated with yeast extract and then cultured in a bioreactor for 80 days. The adventitious roots were collected as industrial hemp adventitious roots for conventional scale-up culture.

[0133] The liquid culture medium and fed culture medium used in this experiment had the same composition, both including 3 / 4 MS medium, 2.5 mg / L IBA and 25 g / L sucrose.

[0134] The investigation collected data on the growth of adventitious roots and the accumulation of target metabolites. The results are shown in [the table below]. Figure 8 and Figure 9The adventitious roots after fed-batch culture differentiated into new lateral roots and exhibited significant elongation growth. Their biomass was significantly higher than that of adventitious roots from conventional scale-up culture. Simultaneously, the adventitious roots accumulated higher levels of cannabinoids and tanshinone, with particularly significant increases in tetrahydrotanshinone I, CBG, and CBD. These levels were 3.32 times, 3.99 times, and 8.53 times higher than those of untreated adventitious roots from conventional scale-up culture with yeast extract, respectively, and 2.56 times, 2.84 times, and 3.76 times higher than those of adventitious roots treated with yeast extract from conventional scale-up culture. This demonstrates that the fed-batch culture model not only promotes the growth, proliferation, and differentiation of industrial hemp adventitious roots but also facilitates the accumulation of target metabolites within the adventitious roots.

[0135] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. 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 this application. Therefore, this application 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 claimed in this application.

Claims

1. A method for cultivating adventitious roots of industrial hemp that produce high yields of cannabinoids and tanshinone, characterized in that, Includes the following steps: Yeast extract was used to induce the adventitious roots of industrial hemp, resulting in induced adventitious roots of industrial hemp. A bioreactor was used to scale up the cultivation of adventitious roots of induced industrial hemp. Collect adventitious roots of industrial hemp after large-scale cultivation; The yeast extract is added at a rate of 300 mg to 400 mg per liter of liquid culture medium.

2. The method for cultivating high-yield industrial hemp adventitious roots of cannabinoids and tanshinone according to claim 1, characterized in that, The method of inducing industrial hemp adventitious roots with yeast extract to obtain induced industrial hemp adventitious roots specifically includes: Under aseptic conditions, adventitious roots of industrial hemp were inoculated into shake flasks containing liquid culture medium and cultured to obtain adventitious roots of industrial hemp in suspension. Yeast extract was added to the suspended cultured adventitious roots of industrial hemp and cultured for another 48 to 96 hours to obtain the induced industrial hemp adventitious roots.

3. The method for cultivating adventitious roots of industrial hemp with high yields of cannabinoids and tanshinone according to claim 1, characterized in that, The process of culturing adventitious roots of industrial hemp using suspension culture technology to obtain an industrial hemp adventitious root suspension culture system specifically includes: placing industrial hemp adventitious roots in a shake flask containing liquid culture medium and shaking and incubating in the dark for 30-40 days to obtain an industrial hemp adventitious root suspension culture system.

4. The method for cultivating adventitious roots of industrial hemp with high yields of cannabinoids and tanshinone according to claim 3, characterized in that, The temperature for dark incubation with shaking is 20℃~30℃, and the rotation speed is 90 r / min~110 r / min.

5. The method for cultivating adventitious roots of industrial hemp with high yields of cannabinoids and tanshinone according to claim 1, characterized in that, The process of using a bioreactor to scale up the adventitious roots of induced industrial hemp specifically includes: The induced adventitious roots of industrial hemp were inoculated into liquid culture medium in a bioreactor for the first scale-up culture. The adventitious roots of industrial hemp after the first scale-up culture were inoculated into a bioreactor, and a second scale-up culture was carried out using a fed-batch culture mode.

6. The method for cultivating adventitious roots of industrial hemp with high yields of cannabinoids and tanshinone according to claim 5, characterized in that, During scale-up cultivation, the inoculation density of the adventitious roots of the industrial hemp was 10 g / L–15 g / L.

7. The method for cultivating adventitious roots of industrial hemp with high yields of cannabinoids and tanshinone according to claim 5, characterized in that, The first scale-up culture must meet at least one of the following conditions: the culture temperature is 20℃~30℃, the culture time is 40d~50d, and the aeration rate is 0.06vvm~0.08vvm.

8. The method for cultivating adventitious roots of industrial hemp with high yields of cannabinoids and tanshinone according to claim 5, characterized in that, The second scale-up culture using a fed-batch mode specifically includes: after an initial culture period, adding feed medium to the bioreactor, and then carrying out fed-batch culture. The second amplification culture was conducted at a temperature of 20℃~30℃ with an aeration rate of 0.06 vvm~0.08 vvm. The initial culture time was 45d~55d, and the amount of feed medium added was 25%~35% of the initial culture medium. The feed culture time was 30d~40d.

9. The method for cultivating adventitious roots of industrial hemp with high yields of cannabinoids and tanshinone according to claim 3, 5, or 8, characterized in that, The liquid culture medium and the feed culture medium comprise: 0.75× MS–MS medium, 2 mg / L~3 mg / L IBA and 25 g / L~35 g / L sucrose, with a pH of 5~6.

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