CN111034546A - Breeding method of industrial cannabis with high CBD content - Google Patents
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Abstract
The invention discloses a breeding and seedling raising method of industrial hemp with high CBD content, which comprises the following steps: (1) selection of parent for high CBD content industrial cannabis: taking industrial hemp seeds with the CBD content of more than 1 percent as male parents, and taking the CBD: taking a heterogynic strain hemp DMG245 with THC content ratio higher than 5 as a female parent; (2) cultivation of high CBD content industrial hemp strains: hybridizing the screened parents to obtain an F1 generation; selfing the F1 generation to obtain F2 generation; backcrossing a single plant with the CBD content of more than 1.5 percent selected from the F2 generation with the DMG245 female parent to obtain an F3 generation; selfing the F3 generation to obtain an F4 generation; backcrossing a single plant with the CBD content of more than 1.5 percent selected from the F4 generation with the DMG245 female parent to obtain an F5 generation; and (4) selfing the F5 generation to obtain an F6 generation, namely the industrial hemp strain with the CBD content of more than 1.5 percent.
Description
Technical Field
The invention relates to the technical field of industrial hemp breeding, and particularly relates to a breeding method of high-content CBD industrial hemp.
Background
Cannabis is a 1-year-old herbaceous plant of the genus Cannabis (Cannabiacee) of the family Cannabaceae. Industrial hemp refers to hemp with a tetrahydrocannabinol content of less than 0.3%, also known as hemp (hemp), and can be used as fiber, seed, or medicine. The hemp widely used at home and abroad is industrial hemp. The active ingredients with the highest value in the traditional Chinese medicine of the cannabinol compound are tetrahydrocannabinol and cannabidiol which are isomers, but the active effects are greatly different. Cannabidiol is a non-psychoactive ingredient extracted from cannabis sativa, does not cause physiological dependence, has good tolerance, shows certain therapeutic properties on various diseases such as neurological diseases, epilepsy, tumors, inflammation, liver injury, diabetes, pain and the like, and has increasingly important significance in the field of medicine. In countries where the use of industrial cannabis is permitted, it is actually permitted to use CBD (cannabidiol) which has a medicinal effect, i.e. industrial cannabis. The higher the CBD content in industrial hemp, the higher the value. China's mature industrial hemp seeds with high CBD content are Yunuman No. 7, the CBD content is 1.3% at most, and the average value is 1%.
The crossbreeding includes single cross, compound cross, backcross and other methods. The single cross, i.e. the cross between two varieties (single cross) is expressed by AxB, and its hybrid progeny is called single cross species, and because of its simple and economic operation, it is most widely used in production, and generally mainly utilizes the first generation of hybrid, such as Feng carp and Fushou fish. Backcross, that is, the filial generation is continued to be hybridized with one of the parents to strengthen the breeding method of some parent character in the hybrid generation. Backcross breeding can introduce one or several economic traits of a certain population B into another population A. The attention of backcross filial generation selection is focused on a certain target trait, so that a new variety/line with a target shape is finally bred.
CN109673505A discloses a crossbreeding method of industrial hemp, which takes a hermaphrodite industrial hemp DMG240 as a male parent and a hermaphrodite industrial hemp DMG231 with high cannabidiol content as a female parent to obtain an F1 generation hologynic high cannabidiol industrial hemp hybrid, which is named as China hemp No. 1. The average CBD content of China-hemp No. 1 is 3.60 percent, which is 3-4 times of the CBD content of Yunuman No. 7, and the average THC content is 0.18 percent.
CN109122306A discloses a method for obtaining industrial hemp plants with high cannabidiol content, which comprises the following steps: when the industrial hemp seedling plant grows to the jointing stage or the grouting stage, wrapping or placing a cotton ball on the stem tip of the plant, and then dripping ethyl methane sulfonate solution on the cotton ball on the stem tip of the plant to perform mutagenesis reaction; removing the cotton balls and using NaS2O3Coating the stem tip with the solution to terminate the mutagenesis reaction, and then washing with running water to form a length and height mark; when the industrial hemp plants grow upwards from the marked positions to obtain the growth heights, cutting the cutting shoots, transplanting the cutting shoots after the cutting shoots root, and performing conventional cultivation management; and (3) processing the mutant plants by ethyl methanesulfonate to obtain viable mutant plants, and screening the mutant plants with high cannabidiol CBD content after character stability verification.
However, there is still a need to find new lines of industrial cannabis that increase the CBD content in industrial cannabis flowers.
Disclosure of Invention
The invention provides a breeding method of industrial cannabis with high CBD content, which comprises the following steps:
(1) selection of parent for high CBD content industrial cannabis: taking industrial hemp seeds with the CBD content of more than 1 percent as male parents, and taking the CBD: taking a heterogynic strain hemp DMG245 with THC content ratio higher than 5 as a female parent;
(2) the cultivation of the industrial hemp strain with high CBD content comprises the following specific steps:
hybridizing the screened parents to obtain an F1 generation;
selfing the F1 generation to obtain F2 generation;
backcrossing a single plant with the CBD content of more than 1.5 percent selected from the F2 generation with the DMG245 female parent to obtain an F3 generation;
selfing the F3 generation to obtain an F4 generation;
backcrossing a single plant with the CBD content of more than 1.5 percent selected from the F4 generation with the DMG245 female parent to obtain an F5 generation;
selfing the F5 generation to obtain F6 generation, and obtaining the hemp strain with stable CBD content of more than 1.5%.
Preferably, the industrial hemp seeds with the CBD content of more than 1 percent are used as male parents, and the industrial hemp Yunyan No. 7 with the CBD content of more than 1 percent is selected as the male parents.
Preferably, the marijuana DMG245 as a male and female heterostrain has the characteristics of disease resistance, insect resistance, low toxicity and salt tolerance.
Preferably, the method for measuring the content of the CBD is to measure the content of the CBD in the industrial cannabis sativa leaves by an HPLC method. More preferably, the chromatographic column in the HPLC method is a C18 column; the HPLC method is characterized in that the mobile phase methanol-water (80: 20); the detection wavelength in the HPLC method is 210 nm; the flow rate of the mobile phase in the HPLC method is 1 ml/min; the sample amount in the HPLC method is 20 μ l.
More preferably, the method for treating industrial hemp leaves in the HPLC method comprises: pulverizing industrial hemp flower and leaf, sieving, weighing 3g, adding methanol 25ml, ultrasonic treating for 15min, cooling, supplementing with methanol, filtering, collecting 3ml filtrate, placing into 10ml measuring flask, and adding methanol to desired volume.
The invention also provides a seedling raising method for the industrial hemp with high CBD content, which comprises the following steps:
(1) obtaining high CBD content industrial hemp seeds: taking industrial hemp seeds with the CBD content of more than 1 percent as male parents, and taking the CBD: taking a heterogynic strain hemp DMG245 with THC content ratio higher than 5 as a female parent; hybridizing the screened parents to obtain an F1 generation; selfing the F1 generation to obtain F2 generation; backcrossing a single plant with the CBD content of more than 1.5 percent selected from the F2 generation with the DMG245 female parent to obtain an F3 generation; selfing the F3 generation to obtain an F4 generation; backcrossing a single plant with the CBD content of more than 1.5 percent selected from the F4 generation with the DMG245 female parent to obtain an F5 generation; selfing the F5 generation to obtain F6 generation, and obtaining the hemp strain with stable CBD content of more than 1.5%.
(2) Filling the seedling substrate into a floating seedling tray;
(3) sowing the industrial hemp seeds obtained in the step (1) in the floating seedling-raising tray obtained in the step (2), and then covering a seedling-raising substrate;
(4) putting the floating seedling-raising plate in the step (3) into a seedling-raising pool;
(5) hardening off seedlings 3-5 days after emergence;
(6) topdressing is carried out on the 9 th to 11 th days of seedling emergence.
Preferably, the seedling culture substrate comprises grass carbon, perlite and vermiculite, and the ratio of the grass carbon, the perlite and the vermiculite is 2-4:1-3: 1; more preferably, the seedling substrate is prepared by mixing turf, perlite and vermiculite according to the ratio of 3:2: 1.
Preferably, the moisture content of the seedling substrate is controlled to be 50% -70% (specifically, 50%, 60%, 70%).
In one embodiment of the invention, the floating seedling tray is a 162-hole floating seedling tray.
In one embodiment of the present invention, the sowing in step (3) comprises sowing 1-5 (e.g., 1, 2, 3, 4, 5) industrial hemp seeds per hole.
Specifically, the thickness of the seedling substrate covered after the seeding in the step (3) is 1-2 cm.
Specifically, the seeds sowed in the step (3) are full seeds which are soaked in warm water and sterilized before sowing.
In one embodiment of the present invention, the warm water soaking treatment comprises: soaking in warm water at 35-45 deg.C for 4-5 h.
In one embodiment of the present invention, the sterilization process comprises: soaking in disinfectant solution for 5-10 min; the bactericide is selected from: one or more of carbendazim, Bordeaux mixture, chlorothalonil, thiophanate and zineb; in one embodiment of the present invention, the bactericide is carbendazim; in one embodiment of the present invention, the bactericide solution is 300-times (e.g., 300, 400, 500, 600, 700-times) carbendazim solution.
Specifically, the step (3) further comprises: sterilizing the surface of the seedling substrate in the seedling tray, such as sterilizing by using a disinfectant solution; the bactericide is selected from: one or more of carbendazim, Bordeaux mixture, chlorothalonil, thiophanate and zineb; in one embodiment of the invention, the bactericide is chlorothalonil; in one embodiment of the present invention, the disinfectant solution is 600-times (specifically, 600, 700, 800, 900, 1000-times) chlorothalonil solution.
In one embodiment of the present invention, the nursery pond in step (4) is previously subjected to water disinfection and disease prevention and fertilization treatment before being placed in the floating nursery tray.
Specifically, the water body disinfection and disease prevention treatment comprises the steps of applying a bactericide solution to the water in the seedling raising pond; the bactericide is selected from: one or more of carbendazim, Bordeaux mixture, chlorothalonil, thiophanate, zineb, fenaminosulf, hymexazol, etc.; in one embodiment of the invention, the germicides include carbendazim and fenaminosulf.
In one embodiment of the invention, the water disinfection and disease prevention treatment comprises applying 75% carbendazim and fenaminosulf to the nursery pond water.
Specifically, the application amount of the 75% carbendazim is 0.1-0.2 g/L.
Specifically, the application amount of the fenaminosulf is 0.1-0.2 g/L.
Specifically, the fertilization treatment comprises the steps of applying a compound fertilizer into the water of the seedling raising pond, wherein the application amount of the compound fertilizer is 0.2-0.35 g/L; in one embodiment of the invention, the ratio of nitrogen, phosphorus and potassium in the compound fertilizer is N: P: K ═ 19:19: 19.
Specifically, the depth of the pond water in the step (4) can be 10-12 cm.
Specifically, the water in the seedling raising pond in the step (4) can be tap water or well water.
Specifically, the seedling raising pond in the step (4) can adopt a black film as a pond film.
Specifically, the nursery pond in the step (4) is positioned under a sun-shading condition, such as under a sun-shading net.
In one embodiment of the present invention, the nursery pond in the step (4) may be located in a nursery greenhouse; specifically, the seedling raising shed adopts a white plastic film as a top film, and a sunshade net is covered on the top film, wherein the sunshade rate of the sunshade net can be 75%.
In one embodiment of the invention, the pond membrane of the seedling raising pond is 7.8m long and 2.2m wide and is fixed along the clamping grooves on the periphery of the seedling raising shed, and the seedling raising shed is 6.0m long, 1.4m wide and 1.2m high.
Specifically, the seedling exercising in the step (5) includes a step of removing the sunshade conditions (such as the sunshade net) before 10 am and after 5 pm, and further includes a step of completely removing the sunshade conditions (such as the sunshade net) after 3 days.
Specifically, the additional fertilizer in the step (6) is to apply compound fertilizer, monopotassium phosphate and carbon energy water soluble fertilizer to the water of the seedling raising pond.
Specifically, the application amount of the compound fertilizer is 0.15-0.25 g/L.
In one embodiment of the invention, the ratio of nitrogen, phosphorus and potassium in the compound fertilizer is N: P: K ═ 30:10: 15.
Specifically, the application amount of the monopotassium phosphate is 0.1-0.2 g/L.
Specifically, the application amount of the carbon energy water-soluble fertilizer is 0.1-0.2 g/L.
In the seedling raising process of the method, the seedling raising environment is ventilated, and the temperature is controlled within 30 ℃.
And (4) 18-20 days after seedling emergence, the plant height of the seedlings reaches 12-15cm, and the whole seedling raising period is completed when root systems are in a bulk shape.
The breeding method accurately selects the industrial hemp seed parents with high CBD content, shortens the breeding period, improves the breeding effect, and ensures that the CBD content in the leaves of the finally hybridized industrial hemp seeds after development can stably reach more than 1.5 percent. The breeding method provided by the invention is less affected by natural climate change, the environment is controllable, the cultivated seedlings are uniform, and the problems of low emergence rate, irregular emergence and the like in field seed direct sowing are effectively solved.
Detailed Description
Example 1 method for measuring CBD content in industrial hemp flower leaves
Preparation of a reference solution: taking appropriate amount of CBD reference substance, adding methanol to obtain reference substance solution containing 0.002, 0.02, 0.2, 1 mg/ml.
Preparation of a sample solution: pulverizing industrial hemp flower and leaf, sieving, weighing 3g, adding methanol 25ml, ultrasonic treating for 15min, cooling, supplementing with methanol, filtering, collecting 3ml filtrate, placing into 10ml measuring flask, and adding methanol to desired volume.
HPLC conditions: chromatography column C18Columns (3.9 mm. times.300 mm, 10 um); mobile phase methanol-water (80: 20); the detection wavelength is 210 nm; the flow rate is 1 ml/min; the amount of the sample was 20. mu.l.
Linear experiments: taking 10 mu 1 of each reference solution, taking the sample amount as the abscissa and the corresponding peak area A as the ordinate to perform linear regression, and the result shows that the linear relation is good in the range of 0.02-0.2 mu g.
Reproducibility and precision experiments: and taking the reference substance solution, continuously injecting samples for 6 times, and performing a precision test. Another batch of industrial hemp flower and leaf samples 6 parts are taken to prepare a sample solution for a reproducibility test. The result shows that the HPLC content determination method has better precision and reproducibility RSD.
And (3) sample determination: preparing a sample solution according to the preparation method of the sample solution, taking 10 mu 1 of sample injection, and calculating the content of CBD in the sample.
Example 2 cultivation of high CBD content Industrial hemp lines
The CBD content in cannabis sativa leaves was determined using the method of example 1. Selecting Yuma No. 7 with the CBD content of more than 1% as a male parent, and selecting the CBD: performing hybridization by using male and female marijuana DMG245 (provided by China academy of agricultural sciences, and derived from China hemp crop germplasm resource middle-term bank) with THC content ratio higher than 5 as female parent to obtain F1 generation; determining the CBD content in F1 generation hemp flowers by the method of example 1, screening out strains with the CBD content of more than 1.5%, and selfing to obtain F2 generation; determining the CBD content in F2 generation hemp flowers by adopting the method of example 1, and carrying out backcross on strains with the CBD content of more than 1.5 percent and a DMG245 female parent to obtain F3 generation; determining the CBD content in the hemp flowers and leaves of the F3 generation by adopting the method of example 1, screening out a strain with the stable CBD content of more than 1.5% from the F3 generation, and selfing to obtain an F4 generation; determining the CBD content in F4 generation cannabis sativa leaves by the method of example 1, screening out a strain with the stable CBD content of more than 1.5% from the F4 generation, and backcrossing the strain with the DMG245 female parent to obtain an F5 generation; selfing the F5 generation to obtain the F6 generation, and determining the content of CBD in the F6 generation hemp leaves by adopting the method of the example 1.
Repeating the method for 3 times to respectively obtain 3 batches of industrial hemp seeds with high CBD content.
TABLE 1 comparison of CBD content of three-batch male parent and F6 generation industrial hemp seed
| CBD content in flower leaves | Batch 1 | Batch 2 | Batch 3 |
| Male parent | 1.12％ | 1.18％ | 1.13％ |
| Generation F6 | 1.63％ | 1.57％ | 1.66％ |
Example 3 seedling raising of high CBD content Industrial hemp lines
(1) Selecting a place with flat terrain, convenient water source, no pollution to water quality and sunny ventilation, constructing a steel frame seedling raising shed with the length of 6.0m, the width of 1.4m and the height of 1.2m, adopting a white plastic film with the thickness of 10 threads as a top film, and covering 1 layer of sunshade net with 75 percent of sunshade rate on the top film. 36 floating seedling plates can be placed in each seedling shed;
(2) adopting a black film with the length of 7.8m, the width of 2.2m and the thickness of 12 threads as a pond film, fixing the pond film along clamping grooves on the periphery of a seedling shed, and manufacturing a seedling pond with the height of about 20 cm;
(3) injecting tap water or well water with the depth of 10cm, applying 100g of 75% carbendazim and 100g of fenaminosulf to each seedling raising pool, sterilizing the water body and preventing diseases, and applying 200g of compound fertilizer with the ratio of N, P and K being 19:19: 19;
(4) preparing a substrate by using grass carbon, perlite and vermiculite according to the ratio of 3:2:1, and sterilizing by using 800 times of carbendazim solution, wherein the water content of the substrate is controlled to be about 60%;
(5) uniformly filling the prepared matrix into a floating seedling raising plate with 162 holes;
(6) selecting industrial hemp seeds obtained by culturing in batch 2 of the embodiment 2, soaking the industrial hemp seeds in warm water at 40 ℃ for 4-5h, and soaking the industrial hemp seeds in 500 times of carbendazim for 5-10min for seed disinfection treatment; sowing 3 seeds in each hole, and covering a substrate with the thickness of 1-2 cm; placing the sown floating seedling-raising tray into a seedling-raising pool, and spraying 800 times of chlorothalonil on the surface layer of the matrix; putting down the side film of the seedling shed and pressing the surrounding plastic film;
(7) keeping the ventilation in the shed, and controlling the temperature in the shed within 30 ℃; uncovering the sunshade net before 10 am and after 5 pm at the beginning of the 4 th day of seedling emergence, and completely uncovering the sunshade net after 3 d; at the 10 th stage of seedling emergence, 150g of compound fertilizer with the ratio of N, P and K being 30:10:15, 100g of monopotassium phosphate and 0.1-0.2g/L of carbon energy water-soluble fertilizer are applied to a seedling raising pond;
18-20 days after seedling emergence, the plant height of the seedling reaches 12-15cm, when the root system is in a bulk shape, the whole seedling raising period is completed, and the seedling rate reaches 97.5 percent
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A breeding method of industrial cannabis with high CBD content is characterized by comprising the following steps:
(1) selection of parent for high CBD content industrial cannabis: taking industrial hemp seeds with the CBD content of more than 1 percent as male parents, and taking the CBD: taking a heterogynic strain hemp DMG245 with THC content ratio higher than 5 as a female parent;
(2) cultivation of high CBD content industrial hemp strains:
hybridizing the screened parents to obtain an F1 generation;
selfing the F1 generation to obtain F2 generation;
backcrossing a single plant with the CBD content of more than 1.5 percent selected from the F2 generation with the DMG245 female parent to obtain an F3 generation;
selfing the F3 generation to obtain an F4 generation;
backcrossing a single plant with the CBD content of more than 1.5 percent selected from the F4 generation with the DMG245 female parent to obtain an F5 generation;
selfing the F5 generation to obtain F6 generation.
2. A breeding method according to claim 1, characterized in that the high CBD content industrial cannabis is of the industrial cannabis line with a CBD content of more than 1.5%.
3. A breeding method according to claim 1, characterised in that industrial hemp Clerodendron 7 with a CBD content of more than 1% is used as male parent.
4. A method as claimed in claim 1, wherein the CBD content is determined by HPLC.
5. A breeding method according to claim 4, characterized in that the chromatographic column in the HPLC method is a C18 column; the HPLC method is characterized in that the mobile phase methanol-water (80: 20); the detection wavelength in the HPLC method is 210 nm; the flow rate of the mobile phase in the HPLC method is 1 ml/min; the sample amount in the HPLC method is 20 μ l.
6. A method as claimed in claim 4, wherein the method of sample solution processing in the HPLC method comprises: pulverizing industrial hemp flower and leaf, sieving, weighing 3g, adding methanol 25ml, ultrasonic treating for 15min, cooling, supplementing with methanol, filtering, collecting 3ml filtrate, placing into 10ml measuring flask, and adding methanol to desired volume.
7. A method for growing seedlings of industrial hemp with high CBD content, the method comprising:
(1) obtaining high CBD content industrial hemp seeds: taking industrial hemp seeds with the CBD content of more than 1 percent as male parents, and taking the CBD: taking a heterogynic strain hemp DMG245 with THC content ratio higher than 5 as a female parent; hybridizing the screened parents to obtain an F1 generation; selfing the F1 generation to obtain F2 generation; backcrossing a single plant with the CBD content of more than 1.5 percent selected from the F2 generation with the DMG245 female parent to obtain an F3 generation; selfing the F3 generation to obtain an F4 generation; backcrossing a single plant with the CBD content of more than 1.5 percent selected from the F4 generation with the DMG245 female parent to obtain an F5 generation; selfing the F5 generation to obtain F6 generation, namely obtaining the hemp strain with stable CBD content of more than 1.5 percent;
(2) filling the seedling substrate into a floating seedling tray;
(3) sowing the industrial hemp seeds obtained in the step (1) in the floating seedling-raising tray obtained in the step (2), and then covering a seedling-raising substrate;
(4) putting the floating seedling-raising plate in the step (3) into a seedling-raising pool;
(5) hardening off seedlings 3-5 days after emergence;
(6) topdressing is carried out on the 9 th to 11 th days of seedling emergence.
8. A breeding method as claimed in claim 7, wherein the seedling substrate comprises peat, perlite and vermiculite in a ratio of 2-4:1-3: 1.
9. A breeding method according to claim 7, characterized in that the floating seedling raising tray is a 162-hole floating seedling raising tray.
10. A breeding method according to claim 7, characterized in that the seeds sown in step (3) are filled seeds that have been warm water soaked and disinfected before sowing.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112997877A (en) * | 2021-02-25 | 2021-06-22 | 黑龙江省科学院大庆分院 | Method for constructing hermaphrodite hemp recombinant inbred line population |
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