A rapid breeding method for high-CBD industrial hemp

Abstract

This invention relates to the field of crop genetics and breeding technology, and particularly to a rapid breeding method for high-CBD industrial hemp. The invention provides a rapid breeding method for high-CBD industrial hemp, which involves constructing a high-segregation-potential F1 generation base population through high-purity parental selection and reciprocal crosses; using oleic acid combined with methyl jasmonic acid to construct a targeted metabolic stress screening system for active ingredient synthesis; completing only one in vitro targeted screening in the F1 generation to identify the core target genotype; then continuously backcrossing to aggregate the superior traits of the recurrent paternal parents, ultimately selecting a line with high CBD content; furthermore, subsequent backcross generations are completed using greenhouse short-day generation combined with rapid, non-destructive in vivo detection technology, eliminating the need for repeated tissue culture operations; finally, after self-pollination purification, a target line with stable traits is obtained.

Description

Technical Field

[0001] This invention relates to the field of crop genetics and breeding technology, and in particular to a rapid breeding method for high-CBD industrial hemp. Background Technology

[0002] Industrial hemp refers to cannabis varieties with a tetrahydrocannabinol (THC) content of ≤0.3%. The cannabidiol (CBD) within these hemp varieties possesses various medicinal properties, including anti-inflammatory, analgesic, anti-anxiety, and anti-epileptic effects. Therefore, high CBD and low THC are core objectives in the breeding of industrial hemp varieties. The CBD / THC ratio is a key indicator for measuring the medicinal value and compliance of a variety; a higher ratio indicates stronger medicinal development value and lower compliance risk.

[0003] Currently, the breeding of new industrial hemp varieties still relies mainly on traditional hybridization breeding, which has the following prominent drawbacks: First, the breeding cycle is lengthy. Traditional hybridization breeding requires multiple generations of self-pollination purification, hybrid combination screening, and multi-year, multi-location identification. Obtaining stable lines typically takes 6-8 years, resulting in extremely low breeding efficiency and difficulty in quickly meeting market demands. Second, existing targeted screening technologies have shortcomings. Molecular marker-assisted breeding relies on high-end equipment and specialized techniques, leading to high testing costs and making large-scale application difficult. In vitro mutagenesis and multi-generation tissue culture screening technologies are prone to causing somatic cell clonal variations, resulting in phenotypic segregation, ploidy abnormalities, and even excessive THC content, making it difficult to guarantee variety stability. Moreover, multi-generation tissue culture operations are cumbersome and have extremely low throughput. Third, the core indicators of existing varieties are relatively low, making it difficult to meet the needs of high-end medicinal development. Fourth, the breeding process carries high compliance risks. Traditional breeding often involves testing for effective components at harvest time, making it impossible to eliminate individual plants with excessive THC in advance. This can easily lead to the final lines exceeding the national mandatory THC standard during field planting, triggering regulatory risks.

[0004] Therefore, developing a rapid breeding method for low-THC, high-CBD industrial hemp with short breeding cycles, low mutation risk, high screening throughput, strong trait orientation, and guaranteed compliance has become a technical challenge that the industry urgently needs to solve. Summary of the Invention

[0005] The purpose of this invention is to provide a rapid breeding method for high-CBD industrial hemp.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a rapid breeding method for high-CBD industrial hemp, comprising the following steps: (1) Select THC content ≤0.2% and CBD content ≥1% as the female parent, and THC content ≤0.15% and CBD content ≥3% as the male parent; (2) The father and mother are orthogonally and reciprocally crossed to obtain orthogonal F1 generation seeds and reciprocal F1 generation seeds, which are then mixed to construct the F1 generation basic segregating population; (3) Select young explants after sterile germination of F1 generation seeds and inoculate them into primary generation directional screening differentiation medium for primary culture to obtain adventitious shoots; transfer the adventitious shoots into secondary generation directional screening medium for secondary culture to obtain secondary screening adventitious shoots. (4) The adventitious buds that were screened again were inoculated into the rooting medium for rooting culture to obtain candidate lines; (5) After transplanting the candidate lines to the field, their THC and CBD contents were tested. The single plants with THC content ≤0.2%, CBD content ≥4.5%, and CBD / THC ratio ≥20:1 were used as recurrent parents. (6) Using the parent in the cycle as the mother in the cycle and the father in step (1) as the father in the cycle, perform continuous backcrossing until the BC3F1 generation population is obtained; (7) The BC3F1 generation population was purified by self-pollination for two consecutive generations to obtain high CBD industrial hemp.

[0007] Preferably, in step (3), the sterile germination step is as follows: after disinfecting the F1 generation seeds, inoculate them into 1 / 2MS solid culture medium and culture them at 20~30℃ under 12h light / 12h dark conditions until germination.

[0008] Preferably, the disinfection method is as follows: F1 generation seeds are disinfected with 70-80% ethanol for 25-35 seconds, rinsed with sterile water 2-4 times, then disinfected with 1-3% sodium hypochlorite solution for 8-12 minutes, and rinsed with sterile water 4-6 times.

[0009] Preferably, the primary directed screening differentiation medium is: MS medium + 6-benzyladenine 1.0~2.0 mg / L + naphthaleneacetic acid 0.1~0.3 mg / L + sucrose 25~35 g / L + agar 6~8 g / L + oleic acid 0.5~2.0 mg / L + methyl jasmonate 0.1~0.5 mg / L, pH 5.8~6.0; The initial culture conditions are as follows: after culturing in complete darkness at 20-30℃ for 2-4 days, the culture is transferred to a temperature of 20-30℃, with 13-15 hours of light / 9-11 hours of darkness and a light intensity of 2000-3000 lx, and cultured for 20-22 days.

[0010] Preferably, in step (3), the subculture directional screening medium is: MS basal medium + 6-benzyladenine 0.5~1.0 mg / L + naphthaleneacetic acid 0.05~0.15 mg / L + sucrose 25~35 g / L + 6~8 g / L + oleic acid 1.0~3.0 mg / L + methyl jasmonate 0.2~0.8 mg / L, pH 5.8~6.0; The conditions for the subculture were 20-30℃, 13-15h light / 9-11h darkness, light intensity of 2000-3000lx, and culture for 18-22 days.

[0011] Preferably, the number of subcultures is 2 to 3 times; During the primary and secondary culture processes, immature explants and explants that have stopped growing or have turned brown and necrotic are discarded.

[0012] Preferably, in step (4), the rooting medium is 1 / 2MS basal medium + indoleacetic acid 0.5~1.0 mg / L + activated carbon 0.3~0.7 g / L + sucrose 15~25 g / L + agar 6~8 g / L, with a pH of 5.8~6.0; The conditions for rooting culture are 20-30℃, 11-13h light / 11-13h darkness, light intensity of 1500-2000 lx, and a culture period of 13-17 days.

[0013] As a preferred embodiment, in step (6), the screening and breeding method for each backcross offspring includes the following steps: 1) Sow backcrossed seeds in a greenhouse to induce the plants to flower earlier; 2) During the full bloom period, the THC and CBD content of the bracts were tested, and single plants with THC content ≤0.2% and CBD content ≥4.5% were selected as backcross female parents; 3) Artificially pollinate and cultivate the selected backcross female parent with the recurrent male parent planted at the same time to harvest the next generation of backcross seeds; 4) Repeat steps 1) to 3) until the BC3F1 generation population is obtained.

[0014] Preferably, in step 1), the temperature of the greenhouse after sowing is set at 25~28℃, the relative humidity at 60~70%, and the light intensity at 800~1200 μmol / (m²). 2 •s), after emergence, perform 10h light / 14h darkness treatment.

[0015] This invention provides the application of the rapid breeding method described above in the rapid breeding of high-CBD industrial hemp.

[0016] Beneficial effects: This invention provides a rapid breeding method for high-CBD industrial hemp. The method involves constructing a high-segregation-potential F1 generation base population through high-purity parental selection and reciprocal crosses. An active ingredient synthesis-directed metabolic stress screening system is constructed using olivine acid and methyl jasmonate. Oligolic acid, as a synthetic precursor, can directionally activate the CBD synthesis pathway, while methyl jasmonate can regulate the expression of key synthetic enzymes. The synergistic effect of these two methods allows for precise screening of genotypes with strong CBD synthesis capacity and inhibited THC synthesis in vitro, significantly reducing the workload of subsequent field screening. This process is completed only once in the F1 generation. In vitro directional screening identifies the core target genotype, fundamentally reducing the risk of somatic cell clonal variation and ploidy abnormalities, ensuring the stable inheritance of the target trait. Then, continuous backcrossing aggregates the superior traits such as stress resistance and high pollen viability of the recurrent paternal parents, ultimately resulting in a strain with high CBD content, uniform plant type, moderate branching, and consistent flowering period, suitable for large-scale standardized planting in my country's main production areas. Furthermore, subsequent backcrossing generations utilize greenhouse short-day generation combined with rapid, non-destructive in vivo detection technology to complete continuous backcrossing, eliminating the need for repeated tissue culture operations. Finally, self-pollination purification yields a trait-stable target strain. This invention shortens the breeding cycle of new industrial hemp varieties from the traditional 6-8 years to 1.5-2 years, significantly reduces the risk of somatic clonal mutations, increases screening throughput by more than 20 times, and significantly reduces breeding costs. The bred strains have a stable tetrahydrocannabinol (THC) content of ≤0.2%, a cannabidiol (CBD) content of ≥5.0%, and a stable CBD / THC ratio of ≥20:1, fully complying with my country's industrial hemp regulatory standards. They also possess high medicinal value and excellent agronomic traits, making them suitable for large-scale commercial breeding and cultivation applications. This invention requires no high-end molecular biology equipment or complex gene editing operations. Conventional seed companies and breeding institutions can quickly master and apply it on a large scale. It combines technological innovation with industrial practicality and can rapidly promote the upgrading of industrial hemp varieties and the high-quality development of the industry in my country. Detailed Implementation

[0017] This invention provides a rapid breeding method for high-CBD industrial hemp, comprising the following steps: (1) Select THC content ≤0.2% and CBD content ≥1% as the female parent, and THC content ≤0.15% and CBD content ≥3% as the male parent; (2) The father and mother are orthogonally and reciprocally crossed to obtain orthogonal F1 generation seeds and reciprocal F1 generation seeds, which are then mixed to construct the F1 generation basic segregating population; (3) Select young explants (cotyledon nodes of sterile seedlings with fully expanded cotyledons and no true leaves) after sterile germination of F1 generation seeds and inoculate them into the primary generation directional screening differentiation medium for primary culture to obtain adventitious buds; transfer the adventitious buds into the secondary generation directional screening medium for secondary culture to obtain secondary screening adventitious buds. (4) The adventitious buds that were screened again were inoculated into the rooting medium for rooting culture to obtain candidate lines; (5) After transplanting the candidate lines to the field, their THC and CBD contents were tested. The single plants with THC content ≤0.2%, CBD content ≥4.5%, and CBD / THC ratio ≥20:1 were used as recurrent parents. (6) Using the parent in the cycle as the mother in the cycle and the father in step (1) as the father in the cycle, perform continuous backcrossing until the BC3F1 generation population is obtained; (7) The BC3F1 generation population was purified by self-pollination for two consecutive generations to obtain high CBD industrial hemp.

[0018] In this invention, both the maternal and paternal parents are industrial hemp strains that have been purified through self-pollination for more than two consecutive generations.

[0019] In this invention, in step (2), the seeds of the male and female parents need to be pre-treated in a directional manner before sowing; the flowering period is controlled by directional cultivation from the budding stage to the initial flowering stage to ensure that the flowering periods of the parents meet completely, and at the same time strengthen the stable expression of the synthetic traits of the parents. The directional pretreatment includes gradient low-temperature stratification and mixed regulator soaking treatment. Specifically, the seeds of the male and female parents are placed at 4℃ for 72 hours of medium-low temperature stratification, and then transferred to 2℃ for 24 hours of medium-low temperature stratification to break dormancy. Subsequently, the seeds are soaked in a sterile aqueous solution containing 0.05~0.2 mg / L brassinolide + 0.5~1.5 mg / L methyl jasmonate at 25℃ in the dark for 12 hours to complete the soaking treatment. The concentration of brassinolide is preferably 0.1~0.15 mg / L, more preferably 0.125 mg / L; The targeted cultivation and regulation of flowering period includes photoperiod regulation and foliar spraying regulation. Specifically, during the budding stage of the male and female parents, a short-day photoperiod regulation of 10h light / 14h darkness is adopted, with a light intensity of 800~1200μmol / (m²). 2 (·s), preferably 900~1100 μmol / (m 2 ·s), further preferably 1000 μmol / (m 2 At the same time, spray the leaves once with an aqueous solution containing 0.8~1.2mg / L salicylic acid during the budding stage and the initial flowering stage. The spraying amount is 30~40L / mu, preferably 33~37L / mu, and even more preferably 35L / mu.

[0020] In this invention, in step (2), both the orthogonal and reciprocal crosses are carried out in a 100-mesh or larger isolation net room. Before pollination, all unopened male buds of the female parent are completely removed. During the pollination period, artificial pollination is performed once a day for 3 to 5 days, preferably 4 days. After pollination, all male parent plants are removed, and the seeds are harvested after they are fully mature.

[0021] In this invention, the sterile germination step (3) is as follows: after disinfecting the F1 generation seeds, they are inoculated into 1 / 2MS solid culture medium and cultured at 20~30℃ under 12h light / 12h dark conditions until germination, with the preferred temperature being 25℃.

[0022] In this invention, the disinfection method is as follows: F1 generation seeds are disinfected with 70-80% ethanol for 25-35 seconds, rinsed with sterile water 2-4 times, then disinfected with 1-3% sodium hypochlorite solution for 8-12 minutes, and rinsed with sterile water 4-6 times. The preferred ethanol concentration is 75%; the preferred ethanol disinfection time is 30 seconds; and the preferred number of rinsing times is 3. The concentration of the sodium hypochlorite solution is preferably 2%, the disinfection time with the sodium hypochlorite solution is preferably 10 minutes, and the number of rinsing cycles is preferably 5.

[0023] In this invention, the primary directed screening differentiation medium is: MS medium + 6-benzyladenine 1.0~2.0 mg / L + naphthaleneacetic acid 0.1~0.3 mg / L + sucrose 25~35 g / L + agar 6~8 g / L + oleic acid 0.5~2.0 mg / L + methyl jasmonate 0.1~0.5 mg / L, pH 5.8~6.0; The concentration of 6-benzyladenine is preferably 1.5 mg / L, the concentration of naphthaleneacetic acid is preferably 0.2 mg / L, the concentration of sucrose is preferably 30 g / L, the concentration of agar is preferably 7 g / L, the concentration of oleic acid is preferably 1.25 mg / L, and the concentration of methyl jasmonate is preferably 0.3 mg / L. The initial culture conditions are as follows: after culturing in complete darkness at 20-30℃ for 2-4 days, the culture is transferred to a temperature of 20-30℃, with 13-15 hours of light / 9-11 hours of darkness and a light intensity of 2000-3000 lx, and cultured for 20-22 days. The optimal temperature for complete darkness incubation is 25℃, and the optimal incubation time is 3 days. The optimal temperature for alternating light and dark culture is 25℃, the optimal light duration is 14h light / 10h dark, the optimal light intensity is 2500lx, and the optimal culture time is 21d.

[0024] In this invention, in step (3), the subculture directional screening medium is: MS basal medium + 6-benzyladenine 0.5~1.0 mg / L + naphthaleneacetic acid 0.05~0.15 mg / L + sucrose 25~35 g / L + 6~8 g / L + oleic acid 1.0~3.0 mg / L + methyl jasmonate 0.2~0.8 mg / L, pH 5.8~6.0; The concentration of 6-benzyladenine is preferably 0.75 mg / L, the concentration of naphthaleneacetic acid is preferably 0.1 mg / L, the concentration of sucrose is preferably 30 g / L, the concentration of agar is preferably 7 g / L, the concentration of oleic acid is preferably 2 mg / L, and the concentration of methyl jasmonate is preferably 0.5 mg / L. The conditions for the subculture were 20-30℃, 13-15h light / 9-11h darkness, light intensity of 2000-3000lx, and culture for 18-22 days. The preferred temperature for the subculture is 25°C, the preferred light duration is 14 hours of light / 10 hours of darkness, the preferred light intensity is 2500 lx, and the preferred culture time is 20 days.

[0025] In this invention, the number of subcultures is 2 to 3 times, preferably 3 times.

[0026] During the primary and secondary culture processes, immature explants and explants that have stopped growing or have turned brown and necrotic are discarded.

[0027] In this invention, in step (4), the rooting medium is 1 / 2MS basal medium + indoleacetic acid 0.5~1.0mg / L + activated carbon 0.3~0.7g / L + sucrose 15~25g / L + agar 6~8g / L, pH value 5.8~6.0; The concentration of indoleacetic acid is preferably 0.75 mg / L, the concentration of activated carbon is preferably 0.5 g / L, the concentration of sucrose is preferably 20 g / L, and the concentration of agar is preferably 7 g / L. The conditions for rooting culture are 20-30℃, 11-13h light / 11-13h darkness, light intensity of 1500-2000lx, and culture period of 13-17 days; The preferred temperature for rooting culture is 25℃, the preferred light duration is 12h light / 12h dark, the preferred light intensity is 1750lx, and the preferred culture time is 15d.

[0028] In this invention, step (6) involves the following steps for screening and breeding offspring from each backcross: 1) Sow backcrossed seeds in a greenhouse to induce the plants to flower earlier; 2) During the full bloom period, the THC and CBD content of the bracts were tested, and single plants with THC content ≤0.2% and CBD content ≥4.5% were selected as backcross female parents; 3) Artificially pollinate and cultivate the selected backcross female parent with the recurrent male parent planted at the same time to harvest the next generation of backcross seeds; 4) Repeat steps 1) to 3) until the BC3F1 generation population is obtained.

[0029] In this invention, in step 1), the temperature of the greenhouse after sowing is set at 25~28℃, preferably 26℃, the relative humidity is 60~70%, preferably 65%, and the light intensity is 800~1200 μmol / (m²). 2 (·s), preferably 900~1100 μmol / (m 2 ·s), further preferably 1000 μmol / (m 2 •s) After emergence, perform 10 hours of light / 14 hours of darkness treatment; The artificial pollination process involves targeted cultivation and control of the flowering period from the budding stage to the initial flowering stage to ensure that the flowering periods of the parent plants coincide.

[0030] This invention provides the application of the rapid breeding method described above in the rapid breeding of high-CBD industrial hemp.

[0031] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] In the embodiments and comparative examples of the present invention, experiments were conducted at the Anda Closed Breeding Base of the Daqing Branch of the Heilongjiang Academy of Agricultural Sciences. Example 1: A rapid breeding method for high-CBD industrial hemp (1) Select industrial hemp lines that have been purified through self-pollination for more than two consecutive generations as parental materials. The female parent is Yunma No. 7 with THC content ≤0.2% and CBD content ≥1%, and the male parent is Zhonghanma No. 1 with THC content ≤0.15% and CBD content ≥3%. (2) The seeds of the male and female parents were placed in a low-temperature stratification environment at 4℃ for 72 hours, and then transferred to a low-temperature stratification environment at 2℃ for 24 hours to break dormancy. Subsequently, the seeds were soaked in a sterile aqueous solution containing 0.1 mg / L brassinolide + 1 mg / L methyl jasmonate at 25℃ in the dark for 12 hours, drained, and then sown. During the budding stage of the male and female parents, a short-day photoperiod of 10 hours of light / 14 hours of darkness was used, with a light intensity of 1000 μmol / (m²). 2At the same time, spray the leaves once with an aqueous solution containing 1.0 mg / L salicylic acid during the budding stage and the initial flowering stage, with a spraying amount of 35 L / mu, to ensure that the flowering periods of the male and female parents meet completely, and at the same time strengthen the stable expression of synthetic traits. The male and female parents were planted in a 120-mesh isolation net room and crosses were carried out in orthogonal and reciprocal directions, respectively. Before pollination, all unopened male buds of the female parent were completely removed to prevent self-pollination. During the pollination period, artificial pollination was carried out once a day from 9 to 10 am for 4 consecutive days. After pollination, all male parent plants were completely removed. After the seeds were fully mature, the F1 generation seeds of the orthogonal cross and the F1 generation seeds of the reciprocal cross were harvested and mixed to construct the F1 generation basic segregating population. (3) F1 generation seeds were disinfected with 75% ethanol for 30 seconds, rinsed 3 times with sterile water, then disinfected with 2% sodium hypochlorite solution for 10 minutes, and rinsed 5 times with sterile water; then inoculated into 1 / 2 MS solid medium and cultured at 25℃ under 12h light / 12h dark conditions until germination. Young explants (cotyledon nodes of sterile seedlings with fully expanded cotyledons and no true leaves) after sterile germination of F1 generation seeds were selected and inoculated into primary generation directed selection differentiation medium (MS medium + 1.5mg / L of 6-benzyladenine + 0.2mg / L of naphthaleneacetic acid + 30g / L of sucrose + 7g / L of agar + 1.2mg / L of oleic acid + 0.3mg / L of methyl jasmonate, pH 6.0). After culturing in complete darkness at 25℃ for 3 days, they were transferred to 25℃, Explants were cultured for 21 days under a 14-hour light / 10-hour dark cycle and a light intensity of 2500 lx. Explants that were stagnant, browned, or necrotic were discarded, and adventitious shoots with vigorous differentiation and growth were retained. The adventitious shoots were then transferred to a subculture medium for directional selection (MS basal medium + 0.8 mg / L 6-benzyladenine + 0.1 mg / L naphthaleneacetic acid + 30 g / L sucrose + 7 g / L sucrose + 2.0 mg / L oleic acid + 0.5 mg / L methyl jasmonate, pH 6.0) and subcultured for 21 days under a 14-hour light / 10-hour dark cycle and a light intensity of 2500 lx at 25℃. This process was repeated twice. Explants that were stagnant, browned, or necrotic were discarded, and adventitious shoots with vigorous differentiation and growth were retained, resulting in the second-screened adventitious shoots. (4) The re-screened adventitious shoots were inoculated into rooting medium (1 / 2 MS basal medium + indoleacetic acid 0.8 mg / L + activated carbon 0.35 g / L + sucrose 20 g / L + agar 7 g / L, pH 5.9) and cultured for 15 days at 25℃, 12h light / 12h dark, and light intensity of 1800 lx to obtain candidate lines; (5) The candidate lines were transplanted to the field and managed in a conventional field. During the full bloom period, the bracts of the female inflorescences were removed. The contents of THC and CBD were detected by HPLC. Single plants with THC content ≤0.2%, CBD content ≥4.5%, and CBD / THC ratio ≥20:1 were selected as recurrent parents. (6) Using the parent in the cycle as the mother in the cycle and the father in step (1) as the father in the cycle, perform backcrossing for three consecutive generations: 1) Sow the backcrossed seeds in a greenhouse, controlling the temperature at 26℃, relative humidity at 65%, and light intensity at 1000 μmol / (m²). 2 •s); Immediately after emergence, perform 10h light / 14h darkness treatment to induce early flowering; 2) During the full bloom period, the THC and CBD content of the bracts were tested, and single plants with THC content ≤0.2% and CBD content ≥4.5% were selected as backcross female parents; 3) Artificially pollinate and bag the selected backcross female parent with the recurrent male parent planted at the same time. By controlling the flowering period from the budding stage to the initial flowering stage, ensure that the flowering periods of the parents are completely coincident. After pollination, continue to cultivate until the seeds mature and harvest the next generation of backcross seeds. 4) Repeat steps 1) to 3), complete the 3rd generation backcross within 12 months to obtain the BC3F generation 1 population; (7) The BC3F1 generation population was purified by self-pollination for two consecutive generations to obtain high CBD industrial hemp.

[0033] Example 2: A rapid breeding method for high-CBD industrial hemp

[0034] (1) Select industrial hemp lines that have been self-pollinated and purified for more than two consecutive generations as parental materials, with the female parent being Longma No. 5, with a THC content ≤0.2% and a CBD content ≥1%, and the male parent being Zhonghanma No. 1, with a THC content ≤0.15% and a CBD content ≥3%; (2) Seeds of the male and female parents were placed in a low-temperature stratification environment at 4℃ for 72 hours, and then transferred to a low-temperature stratification environment at 2℃ for 24 hours to break dormancy. Subsequently, the seeds were soaked in a sterile aqueous solution containing 0.05 mg / L brassinolide + 0.5 mg / L methyl jasmonate at 25℃ in the dark for 12 hours, drained, and then sown. During the budding stage of the male and female parents, a short-day photoperiod of 10 hours of light / 14 hours of darkness was used, with a light intensity of 800 μmol / (m²). 2 At the same time, spray the leaves once with an aqueous solution containing 0.8 mg / L salicylic acid during the budding stage and the initial flowering stage, with a spraying amount of 40 L / mu, to ensure that the flowering periods of the male and female parents meet completely, and at the same time strengthen the stable expression of synthetic traits. The male and female parents were planted in a 100-mesh isolation net room and crosses were carried out in orthogonal and reciprocal directions, respectively. Before pollination, all unopened male buds of the female parent were completely removed to prevent self-pollination. During the pollination period, artificial pollination was carried out once a day from 9 to 10 am for 3 consecutive days. After pollination, all male parent plants were completely removed. After the seeds were fully mature, the F1 generation seeds of the orthogonal cross and the F1 generation seeds of the reciprocal cross were harvested and mixed to construct the F1 generation basic segregating population. (3) F1 generation seeds were disinfected with 75% ethanol for 30 seconds, rinsed 3 times with sterile water, then disinfected with 2% sodium hypochlorite solution for 10 minutes, and rinsed 5 times with sterile water. Then they were inoculated into 1 / 2 MS solid medium and cultured at 25℃ under 12h light / 12h dark conditions until germination. Young explants (cotyledon nodes of sterile seedlings with fully expanded cotyledons and no true leaves) after sterile germination of F1 generation seeds were selected and inoculated into primary generation directed selection differentiation medium (MS medium + 1.0 mg / L 6-benzyladenine + 0.1 mg / L naphthaleneacetic acid + 30 g / L sucrose + 7 g / L agar + 0.5 g / L oleic acid + 0.1 mg / L methyl jasmonate, pH 5.8). After culturing at 25℃ in complete darkness for 3 days, they were transferred to 25℃, 14h light / 12h dark conditions. Explants were initially cultured for 21 days under a 14-hour light / 10-hour dark cycle and a light intensity of 2500 lx. Explants that were stagnant, browned, or necrotic were discarded, and adventitious shoots with vigorous differentiation and growth were retained. These adventitious shoots were then transferred to a subculture medium for directional selection (MS basal medium + 0.5 mg / L 6-benzyladenine + 0.05 mg / L naphthaleneacetic acid + 30 g / L sucrose + 7 g / L sucrose + 1.0 mg / L oleic acid + 0.2 mg / L methyl jasmonate, pH 5.8) and subcultured for 21 days under a 14-hour light / 10-hour dark cycle and a light intensity of 2500 lx. This process was repeated twice. Explants that were stagnant, browned, or necrotic were discarded, and adventitious shoots with vigorous differentiation and growth were retained, resulting in the second-screened adventitious shoots. (4) The re-screened adventitious shoots were inoculated into rooting medium (1 / 2 MS basal medium + indoleacetic acid 0.5 mg / L + activated carbon 0.5 g / L + sucrose 20 g / L + agar 7 g / L, pH 6.0) and cultured for 15 days at 25℃, 12h light / 12h dark, and light intensity of 1800 lx to obtain candidate lines; (5) The candidate lines were transplanted to the field and managed in a conventional field. During the full bloom period, the bracts of the female inflorescences were removed. The contents of THC and CBD were detected by HPLC. Single plants with THC content ≤0.2%, CBD content ≥4.5%, and CBD / THC ratio ≥20:1 were selected as recurrent parents. (6) Using the parent in the cycle as the mother in the cycle and the father in step (1) as the father in the cycle, perform backcrossing for three consecutive generations: 1) Sow the backcrossed seeds in a greenhouse, controlling the temperature at 25℃, relative humidity at 70%, and light intensity at 800 μmol / (m²). 2 •s); Immediately after emergence, perform 10h light / 14h darkness treatment to induce early flowering; 2) During the full bloom period, the THC and CBD content of the bracts were tested, and single plants with THC content ≤0.2% and CBD content ≥4.5% were selected as backcross female parents; 3) Artificially pollinate and bag the selected backcross female parent with the recurrent male parent planted at the same time. By controlling the flowering period from the budding stage to the initial flowering stage, ensure that the flowering periods of the parents are completely coincident. After pollination, continue to cultivate until the seeds mature and harvest the next generation of backcross seeds. 4) Repeat steps 1) to 3), complete the 3rd generation backcross within 14 months to obtain the BC3F generation 1 population; (7) The BC3F1 generation population was purified by self-pollination for two consecutive generations to obtain high CBD industrial hemp.

[0035] Example 3: A rapid breeding method for high-CBD industrial hemp

[0036] (1) Select industrial hemp lines that have been purified through self-pollination for more than two consecutive generations as parental materials. The female parent is Yunma No. 7 with THC content ≤0.2% and CBD content ≥1%, and the male parent is Zhonghanma No. 1 with THC content ≤0.15% and CBD content ≥3%. (2) Seeds of the male and female parents were placed in a low-temperature stratification environment at 4℃ for 72 hours, and then transferred to a low-temperature stratification environment at 2℃ for 24 hours to break dormancy. Subsequently, the seeds were soaked in a sterile aqueous solution containing 0.2 mg / L brassinolide + 1.5 mg / L methyl jasmonate at 25℃ in the dark for 12 hours, drained, and then sown. During the budding stage of the male and female parents, a short-day photoperiod of 10 hours of light / 14 hours of darkness was used, with a light intensity of 1200 μmol / (m²). 2 At the same time, spray the leaves once with an aqueous solution containing 1.2 mg / L salicylic acid during the budding stage and the initial flowering stage, with a spraying amount of 30 L / mu, to ensure that the flowering periods of the male and female parents meet completely, and at the same time strengthen the stable expression of synthetic traits. The male and female parents were planted in a 120-mesh isolation net room and crosses were carried out in orthogonal and reciprocal directions, respectively. Before pollination, all unopened male buds of the female parent were completely removed to prevent self-pollination. During the pollination period, artificial pollination was carried out once a day from 9 to 10 am for 5 consecutive days. After pollination, all male parent plants were completely removed. After the seeds were fully mature, the F1 generation seeds of the orthogonal cross and the F1 generation seeds of the reciprocal cross were harvested and mixed to construct the F1 generation basic segregating population. (3) F1 generation seeds were disinfected with 75% ethanol for 30 seconds, rinsed 3 times with sterile water, then disinfected with 2% sodium hypochlorite solution for 10 minutes, and rinsed 5 times with sterile water. Then they were inoculated into 1 / 2 MS solid medium and cultured at 25℃ under 12h light / 12h dark conditions until germination. Young explants (cotyledon nodes of sterile seedlings with fully expanded cotyledons and no true leaves) after sterile germination of F1 generation seeds were selected and inoculated into primary generation directed selection differentiation medium (MS medium + 6-benzyladenine 2.0 mg / L + naphthaleneacetic acid 0.3 mg / L + sucrose 30 g / L + agar 7 g / L + oleic acid 2.0 g / L + methyl jasmonate 0.5 mg / L, pH 6.0). After being cultured in complete darkness at 25℃ for 3 days, they were transferred to 25℃, 14h light / 12h dark conditions. Explants were initially cultured for 21 days under a 14-hour light / 10-hour dark cycle and a light intensity of 2500 lx. Explants that were stagnant, browned, or necrotic were discarded, and adventitious shoots with vigorous differentiation and growth were retained. These adventitious shoots were then transferred to a subculture medium for directional selection (MS basal medium + 1.0 mg / L 6-benzyladenine + 0.15 mg / L naphthaleneacetic acid + 30 g / L sucrose + 7 g / L sucrose + 3.0 mg / L oleic acid + 0.8 mg / L methyl jasmonate, pH 6.0) and subcultured for 21 days under a 14-hour light / 10-hour dark cycle and a light intensity of 2500 lx. This process was repeated twice. Explants that were stagnant, browned, or necrotic were discarded, and adventitious shoots with vigorous differentiation and growth were retained, resulting in the second-screened adventitious shoots. (4) The re-screened adventitious shoots were inoculated into rooting medium (1 / 2 MS basal medium + indoleacetic acid 1.0 mg / L + activated carbon 0.5 g / L + sucrose 20 g / L + agar 7 g / L, pH 6.0) and cultured for 15 days at 25℃, 12h light / 12h dark, and light intensity of 1800 lx to obtain candidate lines; (5) The candidate lines were transplanted to the field and managed in a conventional field. During the full bloom period, the bracts of the female inflorescences were removed. The contents of THC and CBD were detected by HPLC. Single plants with THC content ≤0.2%, CBD content ≥4.5%, and CBD / THC ratio ≥20:1 were selected as recurrent parents. (6) Using the parent in the cycle as the mother in the cycle and the father in step (1) as the father in the cycle, perform backcrossing for three consecutive generations: 1) Sow the backcrossed seeds in a greenhouse, controlling the temperature at 28℃, relative humidity at 60%, and light intensity at 1200 μmol / (m²). 2 •s); Immediately after emergence, perform 10h light / 14h darkness treatment to induce early flowering; 2) During the full bloom period, the THC and CBD content of the bracts were tested, and single plants with THC content ≤0.2% and CBD content ≥4.5% were selected as backcross female parents; 3) Artificially pollinate and bag the selected backcross female parent with the recurrent male parent planted at the same time. By controlling the flowering period from the budding stage to the initial flowering stage, ensure that the flowering periods of the parents are completely coincident. After pollination, continue to cultivate until the seeds mature and harvest the next generation of backcross seeds. 4) Repeat steps 1) to 3), complete the 3rd generation backcross within 11 months to obtain the BC3F1 generation population; (7) The BC3F1 generation population was purified by self-pollination for two consecutive generations to obtain high CBD industrial hemp.

[0037] Comparative Example 1: Traditional Industrial Hemp Hybridization Breeding Methods

[0038] Using traditional hybridization breeding techniques, the same parental materials as in Example 1 were used for hybridization to obtain F1 generation seeds. The F1 generation was then self-pollinated to obtain the F2 generation segregating population. This process was repeated for six generations of self-pollination and purification. Each generation underwent field planting and HPLC analysis to screen for low THC and high CBD single plants, ultimately obtaining a stable strain. The total breeding cycle was 7 years. The final strain had a THC content of 0.21%~0.25%, a CBD content of 3.8%~4.2%, and a CBD / THC ratio of 15.2:1~20.0:1. These core indicators were significantly lower than those in Example 1 of this invention, and there was a compliance risk that the THC content was close to the national standard threshold. The breeding cycle was more than three times that of Example 1.

[0039] Comparative Example 2: Repeated Screening Method for Multi-Generation Tissue Culture

[0040] Using the same parental materials as in Example 1, an F1 segregating population was constructed. The F1 generation underwent in vitro primary screening and secondary screening to obtain candidate lines. The in vitro tissue culture screening and field identification process was repeated for each subsequent backcross progeny until the BC3F1 generation. After two more generations of self-pollination, stable lines were obtained, with a total breeding cycle of 3.5 years. The final lines showed obvious phenotypic segregation, with a population uniformity of only 82%, 3.2% of individual plants having a THC content exceeding 0.3%, and a somatic cell mutation rate of 8.7%. There were obvious phenotypic instability and compliance risks, and the breeding cycle was more than twice that of Example 1.

[0041] As can be seen from the above embodiments, the present invention provides a rapid breeding method for high-CBD industrial hemp. This method shortens the breeding cycle of industrial hemp, significantly reduces the risk of somatic cell clonal mutation, increases screening throughput by more than 20 times, significantly reduces breeding costs, and does not require high-end molecular biology equipment or complex gene editing operations.

[0042] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A rapid breeding method for high-CBD industrial hemp, characterized in that, Includes the following steps: (1) Select THC content ≤0.2% and CBD content ≥1% as the female parent, and THC content ≤0.15% and CBD content ≥3% as the male parent; (2) The father and mother are orthogonally and reciprocally crossed to obtain orthogonal F1 generation seeds and reciprocal F1 generation seeds, which are then mixed to construct the F1 generation basic segregating population; (3) Select young explants after sterile germination of F1 generation seeds and inoculate them into primary generation directional screening differentiation medium for primary culture to obtain adventitious shoots; transfer the adventitious shoots into secondary generation directional screening medium for secondary culture to obtain secondary screening adventitious shoots. (4) The adventitious buds that were screened again were inoculated into the rooting medium for rooting culture to obtain candidate lines; (5) After transplanting the candidate lines to the field, their THC and CBD contents were tested. The single plants with THC content ≤0.2%, CBD content ≥4.5%, and CBD / THC ratio ≥20:1 were used as recurrent parents. (6) Using the parent in the cycle as the mother in the cycle and the father in step (1) as the father in the cycle, perform continuous backcrossing until the BC3F1 generation population is obtained; (7) The BC3F1 generation population was purified by self-pollination for two consecutive generations to obtain high CBD industrial hemp.

2. The rapid breeding method according to claim 1, characterized in that, In step (3), the sterile germination step is as follows: after disinfecting the F1 generation seeds, they are inoculated into 1 / 2MS solid culture medium and cultured at 20~30℃ under 12h light / 12h dark conditions until germination.

3. The rapid breeding method according to claim 2, characterized in that, The disinfection method is as follows: F1 generation seeds are disinfected with 70-80% ethanol for 25-35 seconds, rinsed with sterile water 2-4 times, then disinfected with 1-3% sodium hypochlorite solution for 8-12 minutes, and rinsed with sterile water 4-6 times.

4. The rapid breeding method according to claim 1, characterized in that, In step (3), the primary directional screening differentiation medium is: MS medium + 6-benzyladenine 1.0~2.0 mg / L + naphthaleneacetic acid 0.1~0.3 mg / L + sucrose 25~35 g / L + agar 6~8 g / L + oleic acid 0.5~2.0 mg / L + methyl jasmonate 0.1~0.5 mg / L, pH 5.8~6.0; The initial culture conditions are as follows: after culturing in complete darkness at 20-30℃ for 2-4 days, the culture is transferred to a temperature of 20-30℃, with 13-15 hours of light / 9-11 hours of darkness and a light intensity of 2000-3000 lx, and cultured for 20-22 days.

5. The rapid breeding method according to claim 1, characterized in that, In step (3), the subculture directional screening medium is: MS basal medium + 6-benzyladenine 0.5~1.0 mg / L + naphthaleneacetic acid 0.05~0.15 mg / L + sucrose 25~35 g / L + 6~8 g / L agar + oleic acid 1.0~3.0 mg / L + methyl jasmonate 0.2~0.8 mg / L, pH 5.8~6.0; The conditions for the subculture were 20-30℃, 13-15h light / 9-11h darkness, light intensity of 2000-3000lx, and culture for 18-22 days.

6. The rapid breeding method according to claim 5, characterized in that, The number of subcultures is 2 to 3 times; During the primary and secondary culture processes, immature explants and explants that have stopped growing or have turned brown and necrotic are discarded.

7. The rapid breeding method according to claim 1, characterized in that, In step (4), the rooting medium is 1 / 2 MS basal medium + indoleacetic acid 0.5~1.0 mg / L + activated carbon 0.3~0.7 g / L + sucrose 15~25 g / L + agar 6~8 g / L, pH 5.8~6.0; The conditions for rooting culture are 20-30℃, 11-13h light / 11-13h darkness, light intensity of 1500-2000 lx, and a culture period of 13-17 days.

8. The rapid breeding method according to claim 1, characterized in that, In step (6), the screening and breeding methods for each backcross offspring include the following steps: 1) Sow backcrossed seeds in a greenhouse to induce the plants to flower earlier; 2) During the full bloom period, the THC and CBD content of the bracts were tested, and single plants with THC content ≤0.2% and CBD content ≥4.5% were selected as backcross female parents; 3) Artificially pollinate and cultivate the selected backcross female parent with the recurrent male parent planted at the same time to harvest the next generation of backcross seeds; 4) Repeat steps 1) to 3) until the BC3F1 generation population is obtained.

9. The rapid breeding method according to claim 8, characterized in that, In step 1), after sowing, the greenhouse temperature is set at 25-28℃, the relative humidity at 60-70%, and the light intensity at 800-1200 μmol / (m²). 2 •s), after emergence, perform 10h light / 14h darkness treatment.

10. The application of the rapid breeding method according to any one of claims 1 to 9 in the rapid breeding of high-CBD industrial hemp.