CN117016391B

CN117016391B - Method for culturing industrial adventitious roots of cannabis sativa for producing cannabidiol and polysaccharide

Info

Publication number: CN117016391B
Application number: CN202311141683.8A
Authority: CN
Inventors: 付玉杰; 安晓丽; 杨杰; 焦骄; 张谡; 李洪权
Original assignee: Northeast Forestry University
Current assignee: Northeast Forestry University
Priority date: 2023-09-05
Filing date: 2023-09-05
Publication date: 2024-05-03
Anticipated expiration: 2043-09-05
Also published as: CN117016391A

Abstract

The invention discloses a method for culturing and producing cannabidiol and polysaccharide industrial cannabis adventitious roots, which uses industrial cannabis aseptic seedling leaves to directly induce and generate adventitious roots, saves callus induction links, simplifies culture medium components and hormone, shortens culture period and improves adventitious root yield; the industrial adventitious roots of cannabis capable of producing stable CBD and polysaccharide in a large quantity in a short period are cultivated by optimizing the culture conditions of a bioreactor such as sucrose concentration, inoculation density, ventilation rate, dynamic research and the like, so that the problems that the yield of the adventitious roots is low, the CBD and the polysaccharide cannot be detected and the like in the prior art are solved; the industrial hemp adventitious roots are transferred from the culture dish to the bioreactor for amplified culture, the culture process is not limited by seasons, climates and soil conditions, the labor intensity and the production cost are reduced, and the annual rapid culture of adventitious roots capable of producing high CBD and polysaccharide is realized.

Description

Method for culturing industrial adventitious roots of cannabis sativa for producing cannabidiol and polysaccharide

Technical Field

The invention relates to the technical field of plant tissue culture, in particular to a method for culturing and producing industrial cannabis adventitious roots of cannabis diphenol and polysaccharide.

Background

Cannabis (Cannabas satava l.), belonging to the genus cannabis of the family cannabis, is distributed in the middle asia, europe and america, etc., and is one of the oldest annual crops in the world, called a medicinal plant with the best potential for large-scale development and utilization. Because of the rapid development of the pharmaceutical market mainly containing phytocannabinoids, many countries are banning the planting of cannabis varieties including high-yielding cannabinoids, and industrial cannabis is becoming a subject of hot spot research.

CBD is one of the main components in industrial cannabis, has no addiction, has great effects in antiepileptic, nerve-protecting, anti-inflammatory, antibacterial and antioxidant aspects, has been used for treating various diseases, and has the potential to develop excellent antioxidants and antibiotics. The plant polysaccharide has pharmacological activities of regulating intestinal tract, resisting oxidation, resisting tumor, reducing blood sugar and the like, has low toxicity and side effects, and has become a hot spot for product development in the fields of food and medicine. Since industrial hemp planting is limited by law, CBD is severely dependent on industrial hemp raw material extraction and separation for obtaining. However, the accumulation of CBD in artificially planted industrial hemp is affected by many factors such as variety, sex, age, growth environment, cultivation method, etc., which makes the quality and availability of industrial hemp raw materials limited and cannot meet market demands. Thus, it is important to find a new way to obtain CBD and polysaccharide.

The adventitious roots of plant tissue culture can gather bioactive substances in plants and keep all characteristics of original plants, and compared with cell culture inheritance, the adventitious roots are more stable in heredity and safety than Mao Zhuang root culture, and the metabolism and activity of the adventitious roots are relatively more stable and are not limited by seasons, climates and soil conditions. The bioreactor can provide excellent growth environment for plant metabolism, makes target culture grow fast and high quality, realizes fast annual production of adventitious roots, can obtain more secondary metabolic substances and a great amount of adventitious roots, and is applied to production of adventitious roots such as ginseng, rehmannia root, astragalus root and the like. The existing industrial cannabis adventitious root culture process is complex and is in a primary stage, for example, a culture system is established after adventitious roots are induced by using calli of Farag and Kayser, the content of cannabidiol, tetrahydrocannabinolic acid and cannabigerolic acid in the adventitious roots under the system is extremely low, and the production is stopped when the industrial cannabis adventitious roots are cultured for 28 days, and CBD and polysaccharide are not detected, so that the existing culture system is not suitable for culturing industrial cannabis adventitious roots to produce CBD and polysaccharide. Therefore, how to realize large-scale production of CBD and polysaccharide is an urgent technical problem to be solved by improving an industrial hemp adventitious root culture system.

Disclosure of Invention

The present invention aims to provide a method for culturing adventitious roots of industrial cannabis for producing cannabidiol and polysaccharide, thereby solving the aforementioned problems in the prior art.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

An industrial adventitious root method for culturing and producing cannabidiol and polysaccharide, comprising the following steps:

S1, inducing industrial hemp aseptic seedlings: selecting industrial hemp seeds, sterilizing, and inoculating to a solid culture medium to obtain industrial hemp aseptic seedlings;

S2, solid culture of industrial hemp adventitious roots: under aseptic condition, inoculating industrial hemp aseptic seedling explant into an induction culture medium I, and directly inducing and culturing adventitious roots; performing proliferation culture on the induced adventitious roots to obtain proliferated industrial hemp adventitious roots;

S3, suspension culture of industrial hemp adventitious roots: the proliferated industrial hemp adventitious roots are taken and put into a bioreactor for amplification culture;

S4, collecting adventitious roots of the industrial hemp cultured in the bioreactor.

Preferably, the step S1 specifically includes:

selecting industrial hemp seeds with full grains, washing the industrial hemp seeds with tap water, screening out seeds with good quality, sterilizing the seeds in a sterile operation table with 75% ethanol for 20-30s, and cleaning the seeds with sterile water; treating with 1% mercuric chloride for 9-10min, cleaning with sterile water, and sucking water on the seed surface with sterile filter paper;

Inoculating to solid culture medium, and culturing at 25+ -2deg.C for 16/8 hr for 15-20d to obtain industrial hemp aseptic seedling.

Preferably, in step S1, the solid medium comprises MS medium, 30-40g/L sucrose and 7-8g/L agar.

Preferably, in step S2, the industrial cannabis aseptic seedling explants are leaves and root tips.

Preferably, the induction medium I is a medium of 3/4MS-MS medium, IBA of 0.5-1mg/L, sucrose of 30-40g/L and agar of 7-8 g/L.

Preferably, in step S2, the induced adventitious roots are subjected to multiplication culture to obtain the proliferated industrial hemp adventitious roots, which specifically includes: under aseptic condition, the adventitious root with good induction state in the last step is cut into about 1-2cm and inoculated to a proliferation culture medium, wherein the proliferation culture medium comprises 3/4MS-MS culture medium, 1.5-3mg/L IBA, 30-40g/L sucrose and 7-8g/L agar.

Preferably, the pH of the medium is=5-6.

Preferably, the medium in the bioreactor in step S3 comprises 3/4MS-MS medium, 1.5-3mg/LIBA and 25-35g/L sucrose.

Preferably, the inoculation density of adventitious roots in the bioreactor in step S3 is 10-15g/L and the aeration rate is 0.06-0.08vvm.

Further preferably, the dark culture time of adventitious roots in the bioreactor is 40-50d.

The beneficial effects of the invention are as follows:

The invention discloses a method for culturing and producing cannabidiol and polysaccharide industrial cannabis adventitious roots by using a bioreactor, wherein industrial cannabis aseptic seedling leaves are directly induced to generate adventitious roots, so that a callus induction link is saved, culture medium components and hormones are simplified, a culture period is shortened, and the yield of the adventitious roots is improved; the industrial adventitious roots of cannabis capable of producing stable CBD and polysaccharide in a large quantity in a short period are cultivated by optimizing the culture conditions of a bioreactor such as sucrose concentration, inoculation density, ventilation rate, dynamic research and the like, so that the problems that the yield of the adventitious roots is low, the CBD and the polysaccharide cannot be detected and the like in the prior art are solved; the industrial hemp adventitious roots are transferred from the culture dish to the bioreactor for amplified culture, the culture process is not limited by seasons, climates and soil conditions, the labor intensity and the production cost are reduced, and the annual rapid culture of adventitious roots capable of producing high CBD and polysaccharide is realized.

Drawings

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

In order to more clearly illustrate the embodiments of the application or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, and it will be obvious to a person skilled in the art that other drawings can be obtained from these drawings without inventive effort.

FIG. 1 is a schematic view showing the growth state of industrial hemp adventitious roots in a culture dish (a), a shake flask (b) and a bioreactor (c), respectively, in the present invention.

FIG. 2 is a high performance liquid chromatograph for detecting the content of CBD in adventitious roots of industrial cannabis obtained by the cultivation method in example 1, wherein (a) is the detection peak of CBD and CBG standard; (b) is the detection peak of CBD and CBG in adventitious roots of industrial hemp.

FIG. 3 is a schematic representation of the effect of sucrose concentration on industrial adventitious root growth and CBD and polysaccharide accumulation in experimental examples.

FIG. 4 is a schematic representation of the effect of seed density on adventitious root growth and CBD and polysaccharide accumulation in industrial hemp in experimental examples.

Figure 5 is a schematic of the aeration rate affecting industrial cannabis adventitious root growth and CBD and polysaccharide accumulation in experimental examples.

FIG. 6 is a schematic diagram of a dynamic study of the growth of adventitious roots of industrial cannabis in accordance with an embodiment of the present application.

FIG. 7 is a flow chart of the production of cannabidiol and polysaccharide using a bioreactor for culturing adventitious roots of industrial cannabis in accordance with an embodiment of the present application.

Detailed Description

Various embodiments of the application may exist in a range of forms; it should be understood that the description in a range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the application; it is therefore to be understood that the range description has specifically disclosed all possible sub-ranges and individual values within that range. For example, it should be considered that a description of a range 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., as well as single numbers within the range, such as 1, 2, 3, 4, 5, and 6, wherever applicable. In addition, whenever a numerical range is referred to herein, it is meant to include any reference number (fractional or integer) within the indicated range.

In the present application, unless otherwise specified, terms such as "upper" and "lower" are used specifically to refer to the orientation of the drawing in the figures. In addition, in the description of the present specification, the terms "include", "comprising" and the like mean "including but not limited to". Relational terms such as "first" and "second", and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Herein, "and/or" describing an association relationship of an association object means that there may be three relationships, for example, a and/or B, may mean: a alone, a and B together, and B alone. Wherein A, B may be singular or plural. Herein, "at least one" means one or more, and "a plurality" means two or more. "at least one", "at least one" or the like refer to any combination of these items, including any combination of single item(s) or plural items(s). For example, "at least one (individual) of a, b, or c," or "at least one (individual) of a, b, and c," may each represent: a, b, c, a-b (i.e., a and b), a-c, b-c, or a-b-c, wherein a, b, c may be single or multiple, respectively.

Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present application are commercially available or may be prepared by existing methods.

The invention provides a method for culturing and producing industrial cannabis adventitious roots of cannabis diphenol and polysaccharide, which comprises the following steps:

In some embodiments, step S1 specifically includes:

In some embodiments, in step S1, the solid medium comprises MS medium, 30-40g/L sucrose, and 7-8g/L agar.

In some embodiments, in step S2, the industrial cannabis aseptic seedling explants are leaves and root tips.

In some embodiments, the induction medium I is a medium of 3/4MS-MS medium, 0.5-1mg/L IBA, 30-40g/L sucrose and 7-8g/L agar.

In some embodiments, in step S2, the induced adventitious roots are subjected to multiplication culture to obtain the proliferated industrial hemp adventitious roots, which specifically includes: under aseptic condition, the adventitious root with good induction state in the last step is cut into about 1-2cm and inoculated to a proliferation culture medium, wherein the proliferation culture medium comprises 3/4MS-MS culture medium, 1.5-3mg/L IBA, 30-40g/L sucrose and 7-8g/L agar.

In some embodiments, the pH of the medium is = 5-6.

In some embodiments, the medium in the bioreactor in step S3 comprises 3/4MS-MS medium, 1.5-3mg/L IBA, and 25-35g/L sucrose.

In some embodiments, the inoculation density of adventitious roots in the bioreactor in step S3 is 10-15g/L and the aeration rate is 0.06-0.08vvm.

In some embodiments, the dark culture time of adventitious roots in the bioreactor is 40-50d.

The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental procedures, which are not specified in the following examples, are generally determined according to national standards. If the corresponding national standard does not exist, the method is carried out according to the general international standard, the conventional condition or the condition recommended by the manufacturer.

Examples

The embodiment provides a method for culturing industrial adventitious roots of cannabis sativa for producing cannabidiol and polysaccharide, which comprises the following steps:

1.1 Induction of industrial Cannabis aseptic seedlings

Selecting industrial hemp seeds with full grains, washing the industrial hemp seeds with tap water, screening out seeds with good quality, sterilizing the seeds in a sterile operation table with 75% ethanol for 20-30s, and cleaning the seeds with sterile water; treating with 1% mercuric chloride for 9-10min, cleaning with sterile water, sucking water on the surface of seed with sterile filter paper, inoculating to solid culture medium containing MS culture medium, 30-40g/L sucrose and 7-8g/L agar, and culturing at 25+ -2deg.C for 16/8 hr (light/dark) for 15-20d to obtain industrial hemp aseptic seedling with good growth state;

1.2 Industrial Cannabis adventitious root solid culture

In order to screen out the optimal solid culture conditions, the following screening experiments were performed:

(1) Explant type selection

Under aseptic condition, 6-9 industrial hemp aseptic seedling leaves (1X 1 cm), stem segments (1-2 cm) and root tips (1-2 cm) are respectively cut and inoculated into an induction culture medium (pH=5-6) containing 1/2MS-MS culture medium, 0.5-1mg/L IBA, 30-40g/L sucrose and 7-8g/L agar, and the direct induction culture of adventitious roots is carried out, 10 repetitions are carried out at each level, the adventitious roots are collected, and the biomass is investigated.

The results are shown in Table 1, and adventitious roots grow out after inoculating the leaf and root for about 7 days; after 30-40d culture, the adventitious roots grow well, the induction rate of the adventitious roots of the leaves, the stems and the roots reaches 93%, 85% and 98%, the fresh weight reaches 0.44g/L, and the dry weight reaches 0.04g/L. The leaf is more suitable for the induction culture of industrial hemp adventitious roots.

TABLE 1 Effect of explant type on Induction culture of adventitious roots of Industrial Cannabis sativa

Explant type	Induction time (d)	Induction Rate (%)	Fresh weight (g/L)	Dry weight (g/L)
					Blade	7±0.07	98±0.01	0.44±0.03	0.04±0.01
Stem segment	10±0.06	85±0.02	0.23±0.02	0.02±0.01
					Root tip	7±0.09	93±0.01	0.34±0.03	0.03±0.01

Note that: values are expressed as mean ± standard deviation (n=10).

(2) Proliferation medium type selection

To select the best multiplication medium type, the adventitious roots of the previous step are cultivated for 6 generations and then inoculated into B5, MS, 1/2MS and WPM containing media respectively, 1mg/L IBA, 30g/L sucrose and 7g/L agar (pH=5-6) are added to each medium, the culture is performed in dark for 40d under the condition of 25+/-2 ℃, the adventitious roots are collected, and the biomass of the adventitious roots is investigated.

The results are shown in Table 2, and the fresh weight and dry weight of biomass inoculated to adventitious roots in a medium containing MS are 1.71.+ -. 0.01g/L and 0.12.+ -. 0.01g/L, respectively, which are significantly higher than those of adventitious roots cultured in other media. It was demonstrated that MS medium was beneficial for adventitious root growth.

TABLE 2 Effect of culture Medium type on proliferation culture of adventitious roots of Industrial Cannabis sativa

Note that: values are expressed as mean ± standard deviation (n=10).

(3) Salt strength screening

Adventitious roots obtained by induction culture were inoculated into media containing 1/4MS, 1/2MS, 3/4MS and MS, respectively, and 2mg/L IBA, 30g/L sucrose and 8g/L agar (pH=5-6) were added to each of the media, and the culture was performed for 40d in dark at 25.+ -. 2 ℃ to collect adventitious roots, and the biomass of the adventitious roots was investigated.

The results are shown in Table 3, in which the biomass of adventitious roots gradually increased under the conditions of medium concentration from 1/4MS to 3/4 MS. In 3/4MS medium, fresh weight (1.76.+ -. 0.01 g/L) and dry weight (0.22.+ -. 0.02 g/L) of adventitious roots reached the highest, and above 3/4MS biomass was slightly reduced but not significantly changed. Therefore, 3/4MS-MS was selected to be suitable for salt strength during the actual culture.

TABLE 3 influence of salt strength on propagation culture of adventitious roots of industrial cannabis

Salt strength	Fresh weight (g/L)	Dry weight (g/L)
			1/4MS	1.24±0.03	0.26±0.01
1/2MS	1.32±0.02	0.20±0.01
			3/4MS	1.76±0.06	0.24±0.02
MS	1.71±0.01	0.22±0.02

Note that: values are expressed as mean ± standard deviation (n=10).

(4) Hormone type and concentration screening

To increase adventitious root yield, the effects of IBA, naphthalene Acetic Acid (NAA) and Indole Acetic Acid (IAA) hormones on adventitious root growth were evaluated separately, and specific experimental procedures were as follows:

The adventitious roots obtained by induction were inoculated into media containing different hormone species (IBA, NAA and IAA) and concentrations (0.5-4 mg/L), respectively, the media including 3/4MS medium, 30g/L sucrose and 7g/L agar, ph=5, and after dark culture for 30d at 25±2 ℃, the adventitious roots were collected and the biomass of the adventitious roots was investigated.

As a result, the biomass and growth status of adventitious roots treated with IBA were better than those of other auxin-treated groups (NAA and IAA), and the fresh weight (3.26.+ -. 0.01 g/L) and dry weight (0.31.+ -. 0.02 g/L) of adventitious roots were higher than those of the optimal treated groups of 1mg/L NAA and IAA when 2mg/L IBA was added to the medium, as shown in Table 4. Whereas IAA (1 mg/L) and NAA (1 mg/L) had less pronounced promoting effect on adventitious root growth. Therefore, 1.5-3mg/L IBA is selected as a proper hormone condition in the actual culture process.

TABLE 4 influence of hormones on proliferation culture of adventitious roots of industrial cannabis

Note that: values are expressed as mean ± standard deviation (n=10).

In order to further accurately determine the polysaccharide and CBD content in the collected adventitious roots, the polysaccharide and CBD content in the adventitious roots is determined by liquid chromatography, and the specific test method is as follows:

(1) Preparation of the sample to be tested

Freeze-dried adventitious root samples (0.1 g) are immersed in 90% (v/v) methanol according to a solid-to-liquid ratio (1:30), ultrasonically extracted for 30min at 50 ℃, filtered, repeatedly extracted for 3 times, and the filtrates are combined to be used as samples to be tested.

(2) CBD and Cannabigerol (CBG) content determination

And filtering the sample to be detected by using a 0.22 mu m organic filter membrane, and measuring by using a high performance liquid chromatography. The chromatographic column used was a C18 reversed phase chromatographic column (4.6mm.times.250 mm,5 μm) with a wavelength of 225nm. The mobile phase is acetonitrile (A) -water (B), isocratic elution is carried out for 0 to 20min, A:80%. The flow rate of the mobile phase is 1mL/min, and the preparation gradient concentration of the CBD standard substance is 0.02-0.2mg/mL. And (3) constructing a standard curve by analyzing the relation between the standard substance concentration and the peak area, and calculating the content of CBD in adventitious roots. However, no CBG species were detected in adventitious roots.

(3) Polysaccharide content detection

0.5ML of the sample to be tested and 0.5mL of 80% methanol solution are taken and mixed evenly, 1mL of 5% phenol is added, then 5mL of concentrated sulfuric acid is added, after reaction for 30min, the absorbance at 490nm is detected by an ultraviolet spectrophotometer. Glucose is used as a standard, and the content of total polysaccharide in adventitious roots is expressed as milligram glucose (purification is more than or equal to 98) equivalent/g DW sample.

2.1 Verifying the Effect of the culture means on adventitious roots of Industrial Cannabis sativa

To facilitate future mass production of adventitious roots of industrial cannabis, CBD and polysaccharide content in adventitious roots of solid culture, shake flask and bioreactor culture were compared.

Under the aseptic condition, 6-9 industrial hemp adventitious roots are sheared to about 1-2cm and inoculated into a culture dish of 3/4MS-MS culture medium, 1.5-3mg/L IBA, 30-40g/L sucrose and 7-8g/L agar (pH=5-6) for solid culture; inoculating 6-8g/L fresh adventitious roots into a shake flask containing 100-200mL of liquid culture medium, performing shake culture, performing dark culture at 25+ -2deg.C and 90-110r/min for 30-40d, and harvesting adventitious roots; then cutting 4-6g/L fresh adventitious roots to about 1-2cm, placing the fresh adventitious roots into a 3L airlift bioreactor filled with 1-2L liquid culture medium, regulating the aeration rate to 0.05-0.1vvm, and performing dark culture for 30-40d at 25+/-2 ℃.

As shown in Table 5, FIG. 1 and FIG. 2, the adventitious roots grown in the shake flask were found to be yellowish and longer, the surface was free of white "fluff", the dry weight was significantly higher than that of the solid culture, and the content of CBD and polysaccharide in the adventitious roots was also significantly increased after liquid phase test to reach 0.57.+ -. 0.01mg/g DW and 518.81.+ -. 0.01mg/g DW, respectively, from FIG. 1. The adventitious roots cultured in the bioreactor are longer, more bubbles are generated, the shape of the adventitious roots is similar to that of the adventitious roots in a shake flask, and the dry weight of the adventitious roots is increased to 3.21+/-0.01 g/L, which is obviously higher than that of solid culture and shake flask culture. In addition, through liquid phase detection, the adventitious roots in the biological incubator accumulate more CBD and polysaccharide (0.81+/-0.02 mg/g DW and 569.32 +/-0.01 mg/g DW), which is obviously higher than the other two culture modes, so that the biological incubator can culture industrial hemp adventitious roots for efficiently producing the CBD and the polysaccharide.

TABLE 5 results of adventitious root growth and CBD and polysaccharide accumulation in Petri dishes, shake flasks and bioreactors

Note that: values are expressed as mean ± standard deviation (n=5).

2.2 Establishment of the culture System of the Industrial Cannabis adventitious root bioreactor

(1) Screening for sucrose concentration

Under aseptic condition, taking adventitious roots after multiplication culture, carrying out 2-3 generations of multiplication culture in a bioreactor, shearing 4g/L of fresh adventitious roots to about 1-2cm, putting into a 3L airlift bioreactor containing 3/4MS-MS culture medium, 1.5IBA and 10-40g/L of sucrose in 1-2L liquid culture medium (pH=5-6), adjusting the aeration rate to 0.05vvm, carrying out dark culture at 25+/-2 ℃ for 30d, collecting adventitious roots, investigating the biomass of the adventitious roots, and detecting CBD and polysaccharide content in the adventitious roots.

As a result, as shown in FIG. 3, when the sucrose concentration in the medium was gradually increased from 10g/L to 30g/L, the biomass and CBD of adventitious roots and the polysaccharide content were gradually increased; when the sucrose concentration is higher than 30g/L, the biomass of adventitious roots and the CBD and polysaccharide content gradually decrease. So 25-35g/L is selected to be proper sucrose concentration in the actual large-scale cultivation process of industrial hemp adventitious roots, which is more beneficial to the growth of adventitious roots and the accumulation of CBD and polysaccharide content.

(2) Screening inoculation Density

Inoculating 5-20g/L fresh industrial hemp adventitious roots after multiplication culture into a bioreactor containing 3/4MS culture medium, 1.5mg/L IBA and 25g/L sucrose, adjusting aeration rate to 0.05vvm, and collecting adventitious roots for detection when dark culture is carried out for 30 d.

As a result, as shown in FIG. 4, when 5-10g/L adventitious roots were introduced into the bioreactor, the biomass was gradually increased, and the CBD and polysaccharide content were also increased; when the inoculation density is higher than 10g/L, the biomass and CBD of adventitious roots and polysaccharide content gradually decrease. Therefore, 10-15g/L is selected to be suitable for inoculation density in the actual large-scale cultivation of industrial hemp adventitious roots.

(3) Screening for aeration rate

10G/L of fresh industrial hemp adventitious roots after multiplication culture are inoculated into a culture medium containing 3/4MS culture medium, 1.5mg/L IBA and 25g/L sucrose, the aeration rate is respectively adjusted to 0.05-0.125vvm, and the adventitious roots are collected for investigation when the culture is performed for 30d in a dark state.

The results are shown in FIG. 5, where the aeration rate was 0.075vvm, the adventitious root growth state and CBD and polysaccharide content were significantly increased. Therefore, in the actual large-scale cultivation of industrial hemp adventitious roots, 0.06-0.08vvm can be selected as a proper aeration rate.

(4) Dynamic study

10/L of fresh industrial hemp adventitious roots after multiplication culture are inoculated into a culture medium containing 3/4MS culture medium, 1.5mg/L IBA and 25g/L sucrose, the aeration rate is adjusted to 0.06vvm, the culture is respectively dark-cultured for 10-50d, and the adventitious roots in different time periods are collected for investigation.

As a result, as shown in FIG. 6, the biomass and CBD and polysaccharide content were significantly increased when adventitious roots were cultured for 40-50d. Therefore, the culture period can be selected to be 40-50d in the actual large-scale culture process of the industrial hemp adventitious roots.

In conclusion, the culture method provided by the application can improve the success rate of adventitious roots induced by industrial hemp leaves, the culture conditions of the optimized proliferation culture medium are 3/4MS-MS culture medium, IBA with the concentration of 1.5-3mg/L and sucrose with the concentration of 30-40g/L, the yield of adventitious roots can be obviously improved, the growth vigor of the adventitious roots is better, and the culture period is short; in addition, adventitious roots were inoculated with 10-15g/L adventitious roots in a bioreactor containing 3/4MS-MS medium, 1.5-3mg/L IBA and 25-35g/L sucrose liquid medium, aeration rate was set to 0.06-0.08vvm, and when dark cultured for 40-50d, the dry weight of adventitious roots and their CBD and polysaccharide content were increased 8.95 times, 3.47 times and 1.18 times, respectively, as compared with shake flasks. Further, the industrial hemp adventitious roots obtained by adopting the bioreactor culture system can be used for culturing the industrial hemp adventitious roots for efficiently producing CBD and polysaccharide, and the culture mode also has the potential of large-scale production.

Therefore, the application not only can improve the yield of the adventitious roots of the industrial hemp, the accumulation of CBD and polysaccharide, but also successfully realizes the enlarged culture of the adventitious roots of the industrial hemp, provides technical reference for producing the CBD and the polysaccharide by utilizing a plant tissue culture technology, and establishes a new way for solving the difficult problem of low CBD yield of the artificially cultivated industrial hemp.

By adopting the technical scheme disclosed by the invention, the following beneficial effects are obtained:

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which is also intended to be covered by the present invention.

Claims

1. A method for culturing and producing industrial cannabis adventitious roots of cannabis diphenol and polysaccharide, which is characterized by comprising the following steps:

s2, solid culture of industrial hemp adventitious roots: under aseptic condition, inoculating industrial hemp aseptic seedling explant into an induction culture medium I, and directly inducing and culturing adventitious roots; inoculating the induced adventitious roots to a proliferation culture medium for proliferation culture to obtain proliferated industrial hemp adventitious roots;

The induction culture medium I is a culture medium of 3/4MS-MS culture medium, IBA of 0.5-1mg/L, sucrose of 30-40g/L and agar of 7-8 g/L; the proliferation culture medium comprises 3/4MS-MS culture medium, 1.5-3mg/L IBA, 30-40g/L sucrose and 7-8g/L agar;

The culture medium in the bioreactor comprises 3/4MS-MS culture medium, 1.5-3mg/L IBA and 25-35g/L sucrose; the inoculation density of adventitious roots in the bioreactor is 10-15g/L, and the aeration rate is 0.06-0.08vvm;

2. The method for culturing adventitious roots of industrial cannabis for producing cannabidiol and polysaccharide as claimed in claim 1, wherein step S1 specifically comprises:

3. The method for culturing adventitious roots of industrial cannabis for producing cannabidiol and polysaccharide as claimed in claim 1, wherein in step S1, the solid medium comprises MS medium, 30-40g/L sucrose and 7-8g/L agar.

4. The method for culturing adventitious roots of cannabis sativa for producing cannabidiol and polysaccharide as claimed in claim 1, wherein in step S2, the industrial cannabis sativa aseptic seedling explants are leaves and root tips.

5. The method for culturing adventitious roots of industrial cannabis for producing cannabidiol and polysaccharide according to claim 1, wherein in step S2, the induced adventitious roots are subjected to proliferation culture to obtain the proliferated industrial cannabis adventitious roots, specifically comprising: under aseptic condition, adventitious roots with good induction state in the last step are selected to be cut into 1-2cm and inoculated on proliferation culture medium.

6. The method for culturing adventitious roots of industrial cannabis for producing cannabidiol and polysaccharide as claimed in any one of claims 2-5, wherein the pH of the culture medium is comprised between 5 and 6.

7. The method for culturing adventitious roots of industrial cannabis for producing cannabidiol and polysaccharide as claimed in claim 1, wherein the dark culturing time of adventitious roots in the bioreactor is 40-50d.