Epimedium inflorescence tissue culture rapid propagation method

Abstract

This invention relates to the field of plant tissue culture and the breeding of superior varieties of traditional Chinese medicinal materials. This invention discloses a method for rapid propagation of Epimedium inflorescences via tissue culture, comprising the following steps: S1, collecting newly sprouted flower inflorescences of Epimedium in early spring, disinfecting them, and setting them aside; S2, cutting the disinfected inflorescences into 1.1-1.2 cm segments, inoculating them into callus induction medium, and culturing them under conditions of 1200-1300 lux light intensity, 12 h / day photoperiod, and 24℃. This invention ensures uniform seedling quality and stable traits through targeted screening of effective components and monitoring of genetic stability. The optimized culture medium ratios at each stage result in a high proliferation coefficient, robust rooting, and a short propagation cycle, enabling efficient year-round propagation. Standardized seedling hardening and transplanting lead to a high survival rate. This method is technically stable and highly applicable, enabling the mass production of high-flavonoid glycoside, genetically stable, high-quality seedlings, fundamentally solving problems such as germplasm degradation, low propagation coefficient, and uneven quality, providing strong support for the breeding and industrialization of superior varieties of Epimedium.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture and breeding of superior varieties of Chinese medicinal materials, specifically a method for rapid propagation of Epimedium inflorescence tissue culture. Background Technology

[0002] Epimedium is a traditional and precious Chinese medicinal herb, widely distributed and with outstanding medicinal value. It is rich in icariin flavonoids, possessing immunomodulatory, anti-inflammatory, anti-aging, and anti-tumor effects. It can improve conditions such as osteoporosis, cerebral ischemia, and rheumatic pain, making it an important variety in the Chinese medicinal herb industry. Hubei Province, as a major province for Chinese medicinal herb resources, has made epimedium cultivation a local specialty industry. Market demand for high-quality medicinal materials with high flavonoid glycoside content continues to rise, and the content of effective components directly determines the quality of the medicinal material and the economic benefits of the industry.

[0003] The current development of the Epimedium industry faces prominent bottlenecks: First, the germplasm resources are limited, and existing varieties have low flavonoid glycoside content and poor genetic stability, making it difficult to meet pharmacopoeia standards and market demands for high quality; second, traditional cultivation relies on manual labor, resulting in low breeding efficiency, frequent pests and diseases, and limited large-scale and standardized planting; third, the primary processing stage lacks standardization, which easily leads to the loss of effective components and makes it difficult to increase the added value of products.

[0004] Traditional seed and division propagation methods suffer from low propagation coefficients, long cycles, and easy segregation of traits, making it impossible to rapidly propagate high-quality seedlings. Therefore, the development of a rapid propagation technology for Epimedium inflorescence tissue culture is of great practical significance for achieving efficient propagation of superior germplasm, promoting variety selection and simplified cultivation, improving the quality and industrial benefits of medicinal materials, and assisting in the upgrading of the Chinese medicinal materials industry chain. Thus, we propose a rapid propagation method for Epimedium inflorescence tissue culture to solve the above-mentioned problems. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a rapid propagation method for Epimedium inflorescence tissue culture, which solves the problems of low propagation coefficient, long cycle, germplasm degradation, low and uneven content of effective components, poor genetic stability, and difficulty in large-scale production of high-quality seedlings in traditional Epimedium cultivation.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for rapid propagation of Epimedium inflorescences via tissue culture, comprising the following steps:

[0007] S1. Collect the newly sprouted flower inflorescences of Epimedium in early spring, disinfect them, and set them aside for later use.

[0008] S2. Cut the disinfected inflorescence into 1.1-1.2cm segments and inoculate them into callus induction medium. Culture them under the conditions of light intensity of 1200-1300 lux, photoperiod of 12h / day and culture temperature of 24℃. Change the medium every 18 days to induce callus formation.

[0009] S3. The callus tissue was transferred to the adventitious shoot induction medium and cultured under the conditions of light intensity 1200-1300 lux, photoperiod 12h / day, and culture temperature 24℃ to induce the formation of adventitious shoots. At the same time, the total content of icariin flavonoids in the leaves of adventitious shoots was detected by high performance liquid chromatography, and adventitious shoots with a total flavonoid glycoside content ≥0.6% dry weight were screened.

[0010] S4. The adventitious shoots obtained from S3 screening were cut and inoculated into shoot proliferation medium. They were subcultured under the conditions of light intensity 2300 lux, photoperiod 14 h / day, and culture temperature 24℃ to induce the formation of clustered shoots. During the subculture process, the genetic stability of the clustered shoots was detected every 3 generations using ISSR molecular markers. Clustered shoots with genetic similarity ≥98% were selected for subsequent culture.

[0011] S5. Cut off a single robust bud obtained from S4 screening, inoculate it into rooting medium, and culture it under the conditions of light intensity 2300 lux, photoperiod 14h / day and culture temperature 24℃ to form white fibrous roots;

[0012] S6. After hardening off the rooted tissue culture seedlings, transplant them into the substrate.

[0013] Preferably, in step S1, the specific disinfection operation is as follows: the inflorescence is rinsed under tap water for 1.5 hours, drained, and then disinfected by shaking with 75% alcohol for 35-40 seconds, rinsed 3 times with sterile water, disinfected with 0.1% mercuric chloride for 4 minutes, and rinsed 5 times with sterile water.

[0014] Preferably, in step S2, the callus induction culture medium is: MS basal medium 4.74 g / L, 2,4-dichlorophenoxyacetic acid (2,4-D) 2.8-3.2 mg / L, indolebutyric acid (IBA) 0.7-0.8 mg / L, sucrose 25 g / L, carrageenan 8.0 g / L, pH 6.3.

[0015] Preferably, in step S2, the culture medium is changed every 18 days, and the callus growth rate is calculated after two consecutive changes.

[0016] Preferably, in S3 and S4, the formulations of the adventitious bud induction medium and the bud proliferation medium are independently as follows: 4.74 g MS dry powder medium, 0.7-0.8 mg 6-benzylaminopurine (6-BA), 0.7-0.8 mg indolebutyric acid (IBA), 25 g sucrose, 8.0 g carrageenan, and the balance being distilled water, with the pH adjusted to 6.3.

[0017] Preferably, in step S4, the subculture cycle is 25-30 days, and the subculture is repeated 3-5 times.

[0018] Preferably, in step S5, the rooting medium is formulated as follows: 1 / 2 MS basal medium, α-naphthaleneacetic acid (NAA) 0.7-0.8 mg / L, activated carbon 0.28-0.32 g / L, sucrose 25 g / L, agar 6.5 g / L, pH 6.3.

[0019] Preferably, in step S6, the substrate is garden soil:vermiculite:peat soil:humus soil = 1.5:1:2:1.5, and the hardening-off time is 45-55 days.

[0020] Preferably, in step S6, the nursery soil is disinfected with a 550-650 times dilution of 75% chlorothalonil wettable powder before transplanting, and the plants are planted at a spacing of 16-18cm, with a shade net providing 65% shade.

[0021] Preferably, the epimedium is any one of Epimedium wushanense, Epimedium sagittatum, Epimedium koreanum, or Epimedium pubescens.

[0022] Beneficial effects

[0023] This invention provides a method for rapid propagation of Epimedium inflorescences via tissue culture. Compared with existing technologies, it has the following advantages:

[0024] This rapid propagation method for Epimedium inflorescence tissue culture, through targeted screening of the total content of icariin flavonoids at the adventitious bud stage, can stably obtain high-quality buds with consistent and effective component content, ensuring that the medicinal quality of the seedlings meets the requirements of the pharmacopoeia and the market. During the propagation culture, ISSR molecular markers are used for genetic stability monitoring, and materials with high genetic similarity are strictly screened to effectively control somatic cell variation and ensure stable and consistent traits in offspring. This invention optimizes the culture medium formula for each culture stage, with a reasonable hormone ratio, resulting in high proliferation coefficients of clustered buds, high rooting rates, robust root systems, and a short overall propagation cycle, enabling efficient year-round propagation of high-quality seedlings.

[0025] The seedling hardening and transplanting process is standardized, the substrate ratio is scientific, and the planting management is precise. The tissue culture seedlings have a high survival rate after acclimatization. This method is technically stable, has good reproducibility, and is highly applicable. It can rapidly produce high-quality Epimedium seedlings with high flavonoid glycoside content and genetic stability in large quantities, fundamentally solving the industry's pain points such as germplasm degradation, low propagation coefficient, and uneven quality. It provides efficient technical support for the breeding, standardized planting, and industrial development of Epimedium. Attached Figure Description

[0026] Figure 1 This is a flowchart of a rapid propagation method for Epimedium inflorescence tissue culture according to the present invention. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] like Figure 1 As shown:

[0029] A rapid propagation method for Epimedium inflorescence tissue culture includes the following steps:

[0030] S1. Collect the newly sprouted flower inflorescences of Epimedium in early spring. Rinse the inflorescences under running tap water for 1.5 hours. After draining the surface water, disinfect them by shaking with 75% alcohol for 35-40 seconds. Immediately rinse them three times with sterile water. Then disinfect them with 0.1% mercuric chloride solution for 4 minutes and rinse them five times with sterile water to thoroughly remove any residual disinfectant. Set aside for use after disinfection.

[0031] S2. Cut the disinfected Epimedium inflorescence into 1.1-1.2cm segments and accurately inoculate them into the callus induction medium. The culture conditions are set as follows: light intensity 1200-1300 lux, photoperiod 12h / day, culture temperature 24℃. The medium is changed every 18 days. After two consecutive changes, the callus growth rate is counted to induce the formation of dense and well-grown callus.

[0032] The callus induction culture medium formula is as follows: MS basal medium 4.74 g / L, 2,4-dichlorophenoxyacetic acid (2,4-D) 2.8-3.2 mg / L, indolebutyric acid (IBA) 0.7-0.8 mg / L, sucrose 25 g / L, carrageenan 8.0 g / L, and the pH of the culture medium is adjusted to 6.3;

[0033] S3. The healthy callus tissue obtained in S2 was transferred to the adventitious shoot induction medium. The culture conditions were the same as those for callus induction: light intensity 1200-1300 lux, photoperiod 12h / day, and culture temperature 24℃ to induce adventitious shoot formation. After the adventitious shoots grew stably, the total content of icariin flavonoids in the leaves of the adventitious shoots was detected by high performance liquid chromatography. High-quality adventitious shoots with a total flavonoid glycoside content ≥0.6% dry weight were screened out, and inferior shoots with low content of effective components were removed.

[0034] S4. Select high-quality adventitious shoots obtained from S3 screening and inoculate them into shoot proliferation medium. Adjust the culture conditions to: light intensity 2300 lux, photoperiod 14 h / day, and culture temperature 24℃. Perform subculture to induce the formation of vigorous clustered shoots. The subculture cycle is set to 25-30 days, and the subculture is repeated 3-5 times. During the subculture process, the genetic stability of the clustered shoots is detected every 3 generations using ISSR molecular markers. Select clustered shoots with genetic similarity ≥98% for subsequent culture to prevent genetically variant strains from entering the breeding system.

[0035] The formulations of the adventitious bud induction medium and the bud proliferation medium are independent: 4.74 g MS dry powder medium, 0.7-0.8 mg 6-benzylaminopurine (6-BA), 0.7-0.8 mg indolebutyric acid (IBA), 25 g sucrose, 8.0 g carrageenan, and the remainder is distilled water. The pH of the medium is adjusted to 6.3.

[0036] S5. Cut off a single robust bud obtained from the screening in S4 and inoculate it into the rooting medium. The culture conditions are maintained as follows: light intensity 2300 lux, photoperiod 14h / day, culture temperature 24℃. Continue to culture until well-developed white fibrous roots are formed at the base of the bud to obtain complete Epimedium tissue culture seedlings.

[0037] The rooting medium formula is as follows: 1 / 2 MS basic medium, α-naphthaleneacetic acid (NAA) 0.7-0.8 mg / L, activated carbon 0.28-0.32 g / L, sucrose 25 g / L, agar 6.5 g / L, and the pH of the medium is adjusted to 6.3;

[0038] S6. Harden off the rooted Epimedium tissue culture seedlings for 45-55 days. After hardening off, transplant them into a special substrate. Before transplanting, disinfect the nursery soil with a 550-650 times diluted solution of 75% chlorothalonil wettable powder. Control the plant spacing at 16-18cm. After transplanting, set up a shade net with a 65% shading rate to ensure the survival rate of the transplanted tissue culture seedlings.

[0039] The transplanting substrate ratio is: garden soil: vermiculite: peat moss: humus = 1.5:1:2:1.5;

[0040] The epimedium mentioned is any one of Epimedium wushanense, Epimedium sagittatum, Epimedium koreanum, or Epimedium pubescens.

[0041] Example 1:

[0042] A rapid propagation method for Epimedium inflorescence tissue culture includes the following steps:

[0043] S1. Collect the new flower inflorescences of Epimedium brevicornu in early spring, rinse with tap water for 1.5 hours, drain, then disinfect with 75% alcohol for 35 seconds, rinse 3 times with sterile water, then disinfect with 0.1% mercuric chloride for 4 minutes, rinse 5 times with sterile water, and set aside for use after disinfection.

[0044] S2. Cut the inflorescence into 1.1cm segments and inoculate them into callus induction medium (MS basal medium 4.74g / L, 2,4-D 2.8mg / L, IBA 0.7mg / L, sucrose 25g / L, carrageenan 8.0g / L, pH 6.3). Culture at 1200 lux light intensity, 12h / day photoperiod, and 24℃. Change the medium every 18 days, and repeat the induction of callus twice.

[0045] S3. The callus tissue was transferred to adventitious shoot induction medium (MS dry powder medium 4.74g, 6-BA 0.7mg, IBA 0.7mg, sucrose 25g, carrageenan 8.0g, distilled water to volume, pH 6.3) and adventitious shoots were induced under the same culture conditions. Adventitious shoots with a total flavonoid glycoside content ≥0.6% by dry weight were screened by high performance liquid chromatography.

[0046] S4. Cut high-quality adventitious buds and inoculate them into bud proliferation medium (same as adventitious bud induction medium). The light intensity is 2300 lux, the photoperiod is 14 h / day, and the culture is carried out at 24℃. The subculture cycle is 25 days, and the subculture is carried out 3 times. ISSR molecular marker detection is performed every 3 generations, and clustered buds with genetic similarity ≥98% are selected.

[0047] S5. Cut robust buds and inoculate them into rooting medium (1 / 2 MS basal medium, NAA 0.7 mg / L, activated carbon 0.28 g / L, sucrose 25 g / L, agar 6.5 g / L, pH 6.3), and culture under the same conditions until white fibrous roots are formed.

[0048] S6. After 45 days of hardening off, transplant the tissue culture seedlings to a substrate of garden soil:vermiculite:peat soil:humus = 1.5:1:2:1.5. Disinfect the nursery soil with 75% chlorothalonil at a dilution of 650 times. Plant the seedlings at a spacing of 16cm and set up a 65% shade net.

[0049] This method, through targeted screening of the total content of icariin flavonoids in adventitious buds, can stably obtain high-quality buds with consistent and effective component content, ensuring that the medicinal quality of the seedlings meets the requirements of the pharmacopoeia and the market. During the propagation culture, ISSR molecular markers are used for genetic stability monitoring, and materials with high genetic similarity are strictly screened to effectively control somatic cell variation and ensure stable and consistent traits in offspring. This invention optimizes the culture medium formula for each culture stage, with a reasonable hormone ratio, resulting in high proliferation coefficients of clustered buds, high rooting rates, robust root systems, and a short overall propagation cycle, enabling efficient year-round propagation of high-quality seedlings.

[0050] The seedling hardening and transplanting process is standardized, with scientific substrate ratios and precise planting management, resulting in a high survival rate for tissue culture seedlings. This method is technically stable, highly reproducible, and widely applicable, enabling the rapid mass production of high-flavonoid glycoside and genetically stable high-quality Epimedium seedlings. It fundamentally solves industry pain points such as germplasm degradation, low propagation coefficient, and uneven quality, providing efficient technical support for the breeding, standardized cultivation, and industrial development of Epimedium.

[0051] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for rapid propagation of Epimedium inflorescences via tissue culture, characterized in that: Includes the following steps: S1. Collect the newly sprouted flower inflorescences of Epimedium in early spring, disinfect them, and set them aside for later use. S2. Cut the disinfected inflorescence into 1.1-1.2cm segments and inoculate them into callus induction medium. Culture them under the conditions of light intensity of 1200-1300 lux, photoperiod of 12h / day and culture temperature of 24℃. Change the medium every 18 days to induce callus formation. S3. The callus tissue was transferred to the adventitious shoot induction medium and cultured under the conditions of light intensity 1200-1300 lux, photoperiod 12h / day, and culture temperature 24℃ to induce the formation of adventitious shoots. At the same time, the total content of icariin flavonoids in the leaves of adventitious shoots was detected by high performance liquid chromatography, and adventitious shoots with a total flavonoid glycoside content ≥0.6% dry weight were screened. S4. The adventitious shoots obtained from S3 screening were cut and inoculated into shoot proliferation medium. They were subcultured under the conditions of light intensity 2300 lux, photoperiod 14 h / day, and culture temperature 24℃ to induce the formation of clustered shoots. During the subculture process, the genetic stability of the clustered shoots was detected every 3 generations using ISSR molecular markers. Clustered shoots with genetic similarity ≥98% were selected for subsequent culture. S5. Cut off a single robust bud obtained from the S4 screening, inoculate it into rooting medium, and culture it under the conditions of light intensity 2300 lux, photoperiod 14h / day, and culture temperature 24℃ to form white fibrous roots; S6. After hardening off the rooted tissue culture seedlings, transplant them into the substrate.

2. The method for rapid propagation of Epimedium inflorescences by tissue culture according to claim 1, characterized in that: In S1, the specific disinfection operation is as follows: rinse the inflorescence under tap water for 1.5 hours, drain it, and then disinfect it by shaking with 75% alcohol for 35-40 seconds, rinse it with sterile water 3 times, disinfect it with 0.1% mercuric chloride for 4 minutes, and rinse it with sterile water 5 times.

3. The method for rapid propagation of Epimedium inflorescences by tissue culture according to claim 1, characterized in that: In S2, the callus induction culture medium is: MS basal medium 4.74 g / L, 2,4-dichlorophenoxyacetic acid (2,4-D) 2.8-3.2 mg / L, indolebutyric acid (IBA) 0.7-0.8 mg / L, sucrose 25 g / L, carrageenan 8.0 g / L, pH 6.

3.

4. The method for rapid propagation of Epimedium inflorescences by tissue culture according to claim 1, characterized in that: In S2, the culture medium is changed every 18 days, and the callus growth rate is calculated after two consecutive changes.

5. The method for rapid propagation of Epimedium inflorescences by tissue culture according to claim 1, characterized in that: In S3 and S4, the formulations of the adventitious bud induction medium and the bud proliferation medium are independently as follows: 4.74 g MS dry powder medium, 0.7-0.8 mg 6-benzylaminopurine (6-BA), 0.7-0.8 mg indolebutyric acid (IBA), 25 g sucrose, 8.0 g carrageenan, and the balance being distilled water, with the pH adjusted to 6.

3.

6. The method for rapid propagation of Epimedium inflorescences by tissue culture according to claim 1, characterized in that: In S4, the subculture cycle is 25-30 days, and the subculture is repeated 3-5 times.

7. The method for rapid propagation of Epimedium inflorescences by tissue culture according to claim 1, characterized in that: In step S5, the rooting medium is formulated as follows: 1 / 2 MS basal medium, α-naphthaleneacetic acid (NAA) 0.7-0.8 mg / L, activated carbon 0.28-0.32 g / L, sucrose 25 g / L, agar 6.5 g / L, pH 6.

3.

8. The method for rapid propagation of Epimedium inflorescences by tissue culture according to claim 1, characterized in that: In S6, the substrate is garden soil:vermiculite:peat soil:humus soil = 1.5:1:2:1.5, and the hardening-off time is 45-55 days.

9. The method for rapid propagation of Epimedium inflorescences by tissue culture according to claim 1, characterized in that: In S6, before transplanting, the nursery soil is disinfected with a 550-650 times dilution of 75% chlorothalonil wettable powder, and the plants are planted at a spacing of 16-18cm. A shade net with a shading rate of 65% is then erected.

10. The method for rapid propagation of Epimedium inflorescences by tissue culture according to claim 1, characterized in that: The epimedium mentioned is any one of Epimedium wushanense, Epimedium sagittatum, Epimedium koreanum, or Epimedium pubescens.

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