Epimedium seed after-ripening method and epimedium breeding method

Abstract

The present application relates to the technical field of Chinese herbal medicine cultivation, and discloses a post-maturation method for Epimedium seed and a breeding method for Epimedium, which comprises seed embryo morphology post-maturation liquid treatment, morphology post-maturation storage and stratification treatment. The present application realizes physiological post-maturation synchronization by using seed embryo morphology post-maturation liquid and temperature variation combined with growth regulating substance treatment, and the seed embryo rate reaches 93.19%. The post-processed Epimedium seed is used for seedling in post-maturation substrate and seedling substrate, and the germination rate of the seedling can reach 86.72% at most. The present application is based on the development process of Epimedium seed, and multiple technologies are applied in succession from the morphological maturation of young seed embryo after the seed separates from the parent body, the physiological post-maturation of the seed embryo to the breaking of dormancy and the breeding of germination.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of Chinese herbal medicine cultivation, and particularly relates to a post-maturation method of Epimedium seed and an Epimedium breeding method. BACKGROUND

[0002] Epimedium is a traditional Chinese medicine for tonifying kidney yang, strengthening muscles and bones, and dispelling wind and dampness, which is mainly used for treating impotence and premature ejaculation, osteoporosis and other diseases. The herbal medicine base includes Epimedium, Epimedium sagittatum, Epimedium pubescens, Epimedium koreanum and Epimedium wushanense, which are collected from the wild to supply the medicine market. Due to the discovery of the inhibitory effect of Epimedium flavonoids on tumor cells and the listing of related new drugs in recent years, the demand in 2022 increased by more than 10% compared with 2021, and the market demand is more than 50,000 tons per year in recent years, and the wild resources decrease by 20% per year. Epimedium has weak sexual reproductive ability under natural conditions, and the flowering rate and seed setting rate are not more than 10%; in the process of exploring wild to domestication, it is difficult to collect seeds at the same time due to the staggered flowering and fruiting time, and most of the small seeds are lost, and the survival rate of wild plants after transplanting is low. After Epimedium seed matures, there is a process of seed embryo after-ripening and dormancy, and the natural germination rate is low.

[0003] In the prior art, when Epimedium is post-matured, methods such as plant hormone gibberellin or sand mixing and direct low-temperature stratification are used, and the seed germination rate after trial is still too low. SUMMARY

[0004] The application provides an Epimedium seed post-maturation method and an Epimedium breeding method, which solves the problem that in the prior art, when Epimedium is post-matured, plant hormone gibberellin or sand mixing and direct low-temperature stratification are used, and the seed germination rate after trial is still too low.

[0005] An Epimedium seed post-maturation method, specifically comprising the following steps:

[0006] Seed embryo morphological after-ripening liquid treatment: Epimedium seeds are soaked in 300-400 mg / L ethephon for 8-12 h and then cleaned; the soaked seeds are soaked in physiological after-ripening liquid for 3-4 days to obtain Epimedium seeds with catalyzed physiological after-ripening process, and the physiological after-ripening liquid is a nitrogen-phosphorus-potassium-calcium-boron-zinc seed embryo morphological after-ripening liquid, which contains 0.6-0.9 g / L nitrogen, 0.1-0.3 g / L phosphorus, 0.5-0.8 g / L potassium, 0.05-0.2 g / L calcium, 2-5 mg / L boron and 1.5-4.0 mg / L zinc;

[0007] Morphological after-ripening storage: the Epimedium seeds with catalyzed physiological after-ripening process after soaking are disinfected for 1-2 h, and then stored in sand at 15-19℃ for 58-78 days to obtain Epimedium seeds with completed seed embryo morphological after-ripening; wherein the volume ratio of seeds to sand is 4:1;

[0008] S3. stratification treatment: after the seed of Epimedium brevicornum Morren is shaped, the seed is treated with 4-12 mg / L flumetralin for 4-6 hours and 0.02-0.05 mg / L thidiazuron for 4-6 hours, and then is stored in a sand storage medium at 2-5℃ for 90-120 days; the sand storage medium is a mixture of sand and sawdust, and the volume ratio of the seed, the sand and the sawdust is 1:3:2.

[0009] Preferably, the concentration of the physiological after-ripening solution is 1.3-1.6%.

[0010] Preferably, the physiological after-ripening solution is prepared by mixing 1.05 g / L NH4NO3, 1.13 g / L KNO3, 0.26 g / L KH2PO5, 0.22 g / L CaCl2·2H2O, 1.21-1.82 mg / L H3BO3 and 3.51-4.38 mg / L ZnSO4·7H2O, and then adding deionized water to 1 L.

[0011] Preferably, the soaking temperature in the physiological after-ripening solution is 15-20℃.

[0012] Preferably, the sand contains water in an amount of 6-11% by mass during the storage of the after-ripening seed, and the sand is sterilized at 120℃ for 25-40 minutes.

[0013] Preferably, the sand storage medium contains water in an amount of 8-12% by mass during the stratification treatment, and the sand is sterilized at 120℃ for 25-40 minutes.

[0014] The second object of the application is to protect a method for breeding Epimedium brevicornum Morren, which uses the after-ripened seed of Epimedium brevicornum Morren obtained by any one of claims 1-6.

[0015] The obtained after-ripened seed of Epimedium brevicornum Morren is buried in a culture medium for 30-35 days to obtain seedlings of Epimedium brevicornum Morren; the culture medium comprises WPM medium+AC 0.5 g / L+vermiculite 30 g / L+SUGAR 30 g / L+IBA 0.5+IAA 0.4+GA 0.8, and the pH value is 6.0.

[0016] The seedlings of Epimedium brevicornum Morren are transplanted into a seedling raising medium for breeding; the seedling raising medium comprises turf soil: 2 mm particle size vermiculite: 3 mm particle size perlite = 3:5:2 by volume.

[0017] Preferably, the after-ripened seed of Epimedium brevicornum Morren is subjected to explant sterilization before being buried in the culture medium; the specific steps of the explant sterilization are as follows: first, the explant is treated with 75% ethanol for 40-60 seconds, then is treated with 1% hypochlorite solution diluted 8 times for 6-12 minutes, and finally is washed with sterile water for 3-5 times.

[0018] Preferably, the transplanting, first with 20-25 ℃ water cleaning seedlings, then 0.2% potassium permanganate disinfection 40 min, then rinse the seedlings, and transplant into the seedling plug, for breeding.

[0019] Preferably, the seedling substrate is placed in a seedling box, the base of the seedling box uses a 72-hole seedling plug, and the cover of the seedling box is provided with two air windows.

[0020] Compared with the prior art, the present application has the following beneficial effects:

[0021] 1. The present application realizes physiological after-ripening synchronization by using seed embryo morphology after-ripening liquid and temperature variation combined with growth regulating substances, and the seed embryo rate reaches 93.19%. The after-processed epimedium seed is used for seedling in the after-ripening substrate and the seedling substrate, and the germination rate of the seedling can reach 86.72% at most. The present application is based on the development process of epimedium seed, and multiple technologies are applied comprehensively from the morphological maturity of young seed embryo after the seed separates from the mother body, the physiological after-ripening of the seed embryo to the breaking of dormancy and the germination breeding.

[0022] 2. The present application can fully and effectively utilize epimedium seed, and rapidly breed a healthy offspring group with good genetic diversity and relatively uniform plant morphology.

[0023] 3. The bottom of the seed collection bag adopts a water-permeable net structure loaded with matrine and berberine, so that the seeds that have not completed morphological maturity and need physiological after-ripening are protected from diseases and pests.

[0024] 4. The WPM culture medium is supplemented with low-concentration and low-intensity auxin treatment and the application of vermiculite substrate, which is beneficial to the full utilization of nutrients of epimedium from a low-concentration mineral element habitat in a high-altitude area, and avoids the callus and poor water physiological process, thereby directly inducing the development of the root and rapidly forming a complete plant with a connected vascular system. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The structure diagram of the lampshade type epimedium seed collector provided for the embodiment 1 of the present application;

[0026] Figure 2 The fan-shaped semicircular ring plane view of the lampshade type epimedium seed collector before installation provided for the embodiment 1 of the present application.

[0027] MARKS DESCRIPTION:

[0028] 1-outer ring; 2-sponge rubber strip bottom pad; 3-magic male and female adhesive tape; 4-sponge rubber strip; 5-inner ring. DETAILED DESCRIPTION

[0029] The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments. 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.

[0030] Unless otherwise specified, the experimental methods described in the various embodiments of this invention are conventional methods. Unless otherwise specified, the materials and reagents used are commercially available.

[0031] The germplasm sources for this study were collected from Gansu Province, located in the mountainous forest area at the junction of Weiyuan, Longxi, and Xinglong Mountains, at an altitude of 2200 meters. The annual precipitation is nearly 400 mm, the frost-free period is 140 days, and the average annual temperature is 8.5℃. The selection of germplasm sources was based on the following reasons: Firstly, considering the combined prices of Epimedium from the China Medicinal Herbs Network and Yaotong.com, Gansu germplasm sources have the highest prices, the best quality, and the most stable prices, approximately 2-3 times higher than other sources. Secondly, Gansu germplasm resources, especially those obtained from high-altitude, low-rainfall, and cold-climate habitats, exhibit stronger adaptability and can withstand drought and cold stress, allowing for propagation and promotion over a wider area.

[0032] Example 1

[0033] A method for post-ripening of Epimedium seeds, specifically including the following steps:

[0034] S1. Seed Collection: In early June, observe the lower part of the inflorescence capsules as they begin to turn from green to reddish-brown and begin to split open. Immediately remove the seeds from the lampshade-type Epimedium seed collector. Figure 1 Installed on the plant, its plan view is a semi-circular ring made of polypropylene waterproof and breathable membrane, see Figure 2 The system includes an outer ring 1, an inner ring 5, and a fan-shaped surface. The fan-shaped surface is located between the outer ring 1 and the inner ring 5. The outer ring 1 has an arc length of 47.1cm-67.8cm, the inner arc length of 12.56cm-21.84cm, and the fan-shaped surface is 18cm wide. Magical adhesive strips 3 are designed at both ends. A 12.56cm long, 2cm wide, and 2cm thick sponge strip base 2 is attached to the inside of the inner arc. A sponge strip 4 is placed on the inner side of the sponge strip base 2. The semi-circular ring is rolled up, and the magical adhesive strips 3 are attached together. After installation, the sponge strip 4 shrinks into a circle, tightly surrounding the stem of the Epimedium plant to form the bottom of the pot, catching fallen seeds and fruits. Collect seeds and fruits once a week on a sunny afternoon, with the last collection 10 days later.

[0035] S2. The seed, fruit, fallen leaf mixture in the Epimedium seed collector is gathered together, and the fallen leaves, dry branches and other sundries are picked out. The seed is sterilized by soaking in a mixture of the extract of Sophora flavescens, the extract of Cnidium monnieri and the extract of Phellodendron amurense for 0.5 hours, and the sterilized Epimedium seed is obtained. The mixture has a mass concentration of 0.3% Sophora flavescens extract, 0.1% Cnidium monnieri extract and 0.05% Phellodendron amurense extract, and the rest is water.

[0036] S3. Embryo morphological after-ripening liquid treatment: the sterilized Epimedium seed obtained in S2 is soaked in 300 mg / L of ethephon for 8 hours and then cleaned; and then the seed is transferred into physiological after-ripening liquid for 3 days, with a temperature of 20°C during the day and 15°C at night. The embryo morphological after-ripening liquid is a nitrogen-phosphorus-potassium-calcium-boron-zinc physiological after-ripening liquid containing 0.6 g / L of nitrogen, 0.1 g / L of phosphorus, 0.5 g / L of potassium, 0.05 g / L of calcium, 2 mg / L of boron and 1.5 mg / L of zinc. The concentration of the physiological after-ripening liquid is 1.3%, and the preparation method is as follows: 1.05 g / L of NH4NO3 is added to a non-metal container, and then 25% of the final total volume of deionized water is added and stirred until completely dissolved; 1.13 g / L of KNO3 is added, and then 15% of the final total volume of deionized water is added and stirred until completely dissolved; 0.26 g / L of KH2PO5 is added, and then 15% of the final total volume of deionized water is added and stirred until completely dissolved; 0.22 g / L of CaCl2·2H2O is added, and then 20% of the final total volume of deionized water is added and stirred until completely dissolved; 1.21 mg / L of H3BO3 is added, and then 10% of the final total volume of deionized water is added and stirred until completely dissolved; 3.51 mg / L of ZnSO4·7H2O is added, and then 10% of the final total volume of deionized water is added and stirred until completely dissolved; and deionized water is added to make up to 1 L.

[0037] S4. Morphological after-ripening storage: the soaked Epimedium seed obtained in S1 is spread into a thin layer with a thickness of 1 cm on a flat plate, and then sterilized under a UV lamp for 1 hour. Then the seed is stored in sand at 15°C for 58 days to obtain the Epimedium seed with completed embryo morphological after-ripening. The volume ratio of the seed to the sand is 4:1. The sand has a mass ratio of water content of 6% and is sterilized at 120°C for 25 min.

[0038] S5. stratification treatment: the S4 obtained after the completion of embryo form post-mature Epimedium seed screening, remove the rotten seed, ultraviolet irradiation sterilization 1h, respectively, through 4mg / L fluazinam seed treatment 4h and 0.02mg / L thidiazuron seed treatment 4h, in the bottom of the wall thickness 2mm above the tofu transfer basket 645x445x195mm, the seed and sand storage substrate mix, 2.0℃ cold storage 90 days, endosperm and seed coat ABA concentration is less than 1mg / L, the whole seed gibberellin content is more than 2mg / L, then complete stratification treatment. The sand storage substrate is a mixture of sand and sawdust, according to the volume ratio: seed:sand:sawdust = 1:3:2. The sand storage substrate contains 8% of water by mass ratio, and is sterilized at 120℃ for 25min.

[0039] S6. sampling inspection embryo development, to 90% cotyledon stage, then can be sown.

[0040] An Epimedium breeding method, the specific steps are:

[0041] 1). disinfection: the obtained post-mature Epimedium seed explant disinfection, the specific steps are: first with 75% ethanol treatment 40s, then with 1% hypochlorite solution dilution 8 times treatment 6min, finally again with sterile water washing 3 times, get the disinfection of seed.

[0042] 2). the seed after disinfection is inoculated into culture bottle culture medium for 30 days to obtain Epimedium seedling; the culture medium composition is WPM culture medium + AC 0.5g / L + vermiculite 30g / L + sucrose 30g / L + IBA 0.5 + IAA 0.4 + GA 0.8, pH = 6.0.

[0043] 3). after the root length of Epimedium seedling in 2) is 2cm, and 3 true leaves grow out, gradually open the culture bottle cap to acclimate for a week, transplant from the culture bottle to the seedling box, which contains special Epimedium seedling substrate. The substrate preparation volume ratio: grass carbon soil: 2mm particle size vermiculite: 3mm particle size perlite = 3:5:2, 5L of 40mg / L chlorine dioxide solution is sprayed 0.1m 3 substrate, sealed overnight.

[0044] The base of the seedling box is 72-hole seedling hole tray, which contains the substrate described in 3); when transplanting, the seedlings are first washed with 20℃ clean water, then disinfected with 0.2% potassium permanganate for 40min, then washed clean with clean water, a small shallow pit is made in the center of the substrate of the hole tray with a 0.5cm wide bamboo sheet, the seedling is planted, the index finger and middle finger are lightly pressed at the base of the seedling, after spraying water, the cover of the seedling box with air window is covered, and the two air windows on the cover are opened.

[0045] Example 2

[0046] A method for post-ripening of Epimedium seeds, specifically including the following steps:

[0047] S1. Seed Collection: In early June, observe the lower part of the inflorescence capsules as they begin to turn from green to reddish-brown and begin to split open. Immediately remove the seeds from the lampshade-type Epimedium seed collector. Figure 1 Installed on the plant, its plan view is a semi-circular ring made of polypropylene waterproof and breathable membrane, see Figure 2 The system includes an outer ring 1, an inner ring 5, and a fan-shaped surface. The fan-shaped surface is located between the outer ring 1 and the inner ring 5. The outer ring 1 has an arc length of 47.1cm-67.8cm, the inner arc length of 12.56cm-21.84cm, and the fan-shaped surface is 19cm wide. Magical adhesive strips 3 are designed at both ends. A 12.56cm long, 2cm wide, and 2cm thick sponge strip base 2 is attached to the inside of the inner arc. A sponge strip 4 is placed on the inner side of the sponge strip base 2. The semi-circular ring is rolled up, and the magical adhesive strips 3 are attached together. After installation, the sponge strip 4 shrinks into a circle, tightly surrounding the stem of the Epimedium plant to form the bottom of the pot, catching fallen seeds and fruits. Collect seeds and fruits once a week on a sunny afternoon, with the last collection 10 days later.

[0048] S2. The mixture of seeds, fruits, and fallen leaves in the Epimedium seed collector is gathered together. Fallen leaves, withered branches, and other debris are removed. The mixture is then passed through a 5mm mesh sieve to remove impurities such as petals, fruit stalks, withered leaves, and petioles. The seeds are then disinfected by soaking them in a mixture of Sophora flavescens extract, Cnidium monnieri extract, and Phellodendron amurense extract for 0.5 hours. The disinfected Epimedium seeds are obtained. The mixture contains, by mass concentration: 0.3% matrine extract, 0.1% Cnidium monnieri extract, 0.05% Phellodendron amurense extract, with the remainder being water.

[0049] S3. Embryo morphological after-ripening liquid treatment: the disinfected Epimedium seed obtained in S2 is soaked in 350 mg / L of ethephon for 10 h, and then cleaned; and then transferred into physiological after-ripening liquid for treatment for 4 days, with a temperature of 20℃ during the day and 15℃ at night, wherein the embryo morphological after-ripening liquid is a nitrogen-phosphorus-potassium-calcium-boron-zinc physiological after-ripening liquid containing 0.6-0.9 g / L of nitrogen, 0.2 g / L of phosphorus, 0.6 g / L of potassium, 0.12 g / L of calcium, 4 mg / L of boron and 3.0 mg / L of zinc; the concentration of the physiological after-ripening liquid is 1.4%, and the preparation method is as follows: in a non-metal container, 1.05 g / L of NH4NO3 is added first, then 25% of the final total volume of deionized water is added, and then stirred until completely dissolved; 1.13 g / L of KNO3 is added, then 15% of the final total volume of deionized water is added, and then stirred until completely dissolved; 0.26 g / L of KH2PO5 is added, then 15% of the final total volume of deionized water is added, and then stirred until completely dissolved; 0.22 g / L of CaCl2·2H2O is added, then 20% of the final total volume of deionized water is added, and then stirred until completely dissolved; 1.52 mg / L of H3BO3 is added, then 10% of the final total volume of deionized water is added, and then stirred until completely dissolved; 4.08 mg / L of ZnSO4·7H2O is added, then 10% of the final total volume of deionized water is added, and then stirred until completely dissolved; and finally, deionized water is added to make up to 1 L.

[0050] S4. Morphological after-ripening storage: the soaked Epimedium seed obtained in S1 is spread on a flat plate to form a thin layer with a thickness of 1.2 cm, and then disinfected under ultraviolet light for 2 h, and then stored in sand at 17℃ for 68 days to obtain the Epimedium seed with completed embryo morphological after-ripening; wherein the volume ratio of the seed to the sand is 4:1; and the sand contains water in an amount of 9% by mass and is sterilized at 120℃ for 30 min.

[0051] S5. Stratification treatment: the Epimedium seed with completed embryo morphological after-ripening obtained in S4 is screened to remove rotten and deteriorated seeds, and then sterilized by ultraviolet light for 1 h, and then treated by soaking in 8 mg / L of flumetralin for 5 h and 0.04 mg / L of thidiazuron for 5 h, respectively; and then mixed with a sand storage medium in a bean curd transfer basket with a wall thickness of more than 2 mm to form a mixture, and then stored at 3.0℃ for 105 days; and then the ABA concentration of the endosperm and seed coat is lower than 1 mg / L, and the gibberellin content of the whole seed is more than 2 mg / L, and then the stratification treatment is completed. The sand storage medium is a mixture of sand and sawdust, and the volume ratio of the seed to the sand to the sawdust is 1:3:2; and the sand storage medium contains water in an amount of 10% by mass and is sterilized at 120℃ for 35 min.

[0052] S6. Sampling and checking the embryo development status, and then sowing when the cotyledon stage is reached.

[0053] An Epimedium breeding method, and the specific steps are as follows:

[0054] 1). Disinfection: the obtained Epimedium seed explants are disinfected, and the specific steps are as follows: first, 75% ethanol is used for treatment for 50s, then 1% hypochlorite solution is diluted 8 times for treatment for 9min, and finally, sterile water is used for washing for 4 times, to obtain the disinfected seeds.

[0055] 2). The disinfected seeds are inoculated into the culture bottle culture medium for 33 days to obtain Epimedium seedlings; the culture medium is composed of WPM medium+AC 0.5g / L+vermiculite 30g / L+cane sugar 30g / L+IBA 0.5+IAA 0.4+GA 0.8, pH=6.0.

[0056] 3). After the Epimedium seedlings in 2) have a root length of 2cm and grow 3 true leaves, gradually open the culture bottle cap to acclimate for one week, transplant from the culture bottle to the seedling raising box, and the seedling raising box is placed with a special Epimedium seedling raising substrate. The substrate is prepared in a volume ratio of 3:5:2 of grass carbon soil: 2mm particle size vermiculite: 3mm particle size perlite, and 5L of 40mg / L chlorine dioxide solution is sprayed 0.1m 3 Mix the substrate, and seal overnight.

[0057] The seedling raising box base adopts a 72-hole seedling hole tray, and the hole tray is filled with the substrate described in 3); when transplanting, first, wash the seedlings with 23℃ clean water, then disinfect with 0.2% potassium permanganate for 40min, and then wash the seedlings with clean water again, use a 0.5cm wide bamboo sheet to make a small shallow pit in the center of the substrate of the hole tray, plant the seedlings, press the seedlings at the base with the index finger and middle finger, spray water, cover the seedling raising box cover with air windows, and note that the two air windows on the cover should be opened.

[0058] Example 3

[0059] An Epimedium seed after-ripening method, specifically comprising the following steps:

[0060] S1. Seed collection: in early June, when the lower part of the spike in the inflorescence starts to turn from green to reddish brown and starts to crack, install the lampshade type Epimedium seed collector on the plant, see Figure 1 , which is a semicircular ring made of polypropylene waterproof and breathable film, see Figure 2, including outer ring 1, inner ring 5 and sector, sector is located between the outer ring 1 and the inner ring 5, the outer ring 1 arc length 47.1cm-67.8cm, inner arc length 12.56cm-21.84cm, sector width 20cm, both ends edge design magic male and female adhesive tape 3, 18.84cm long, 2cm wide, 3cm thick sponge rubber strip bottom pad 2 is pasted on the inner arc side, sponge rubber strip 4 is arranged on the inner side of sponge rubber strip bottom pad 2. Roll up the semicircular ring piece, the magic male and female adhesive tape 3 is pasted together, after installation, the sponge rubber strip 4 is shrunk into a circle, the stems of epimedium plants are tightly surrounded, the pot bottom is formed, and the falling seeds and fruits are caught. Collect once a week in the sunny afternoon, and the last time is 10 days apart.

[0061] S2. The seed, fruit and fallen leaf mixture in the epimedium seed collector is gathered together, the fallen leaves, branches and other sundries are picked out, the impurities such as petals, fruit stalks, dry leaves and leaf stalks are removed through a 5mm mesh sieve, the seeds are soaked in a mixture of sophora flavescens extract, cnidium monnieri extract and cortex phellodendri extract for 0.5 hours for disinfection, and the disinfected epimedium seeds are obtained. The mixture has a mass concentration of 0.3% sophora flavescens extract, 0.1% cnidium monnieri extract and 0.05% cortex phellodendri extract, and the rest is water.

[0062] S3. Embryo morphological after-ripening liquid treatment: the disinfected epimedium seeds obtained in S2 are soaked in 400mg / L ethephon for 12 hours and cleaned; then transferred into physiological after-ripening liquid for 4 days, with a temperature of 20℃ in the daytime and 15℃ at night, the embryo morphological after-ripening liquid is a nitrogen, phosphorus, potassium, calcium, boron and zinc physiological after-ripening liquid, containing nitrogen 0.9g / L, phosphorus 0.3g / L, potassium 0.8g / L, calcium 0.2g / L, boron 5mg / L and zinc 4.0mg / L; the concentration of the physiological after-ripening liquid is 1.6%, and the preparation method is as follows: in a non-metal container, 1.05g / L NH4NO3 is added first, then the final total volume is 25% of deionized water, non-metal stirring until completely dissolved, 1.13g / L KNO3 is added, then the final total volume is 15% of deionized water, non-metal stirring until completely dissolved, 0.26g / L KH2PO5 is added, then the final total volume is 15% of deionized water, non-metal stirring until completely dissolved, 0.22g / L CaCl2·2H2O is added, then the final total volume is 20% of deionized water, non-metal stirring until completely dissolved, 1.82mg / L H3BO3 is added, then the final total volume is 10% of deionized water, stirring until completely dissolved, 4.38mg / L ZnSO4·7H2O is added, then the final total volume is 10% of deionized water, stirring until completely dissolved, and then deionized water is added to 1L.

[0063] S4. Morphological after-ripening storage: the soaked Epimedium brevicornum seeds obtained in S1 are spread on a flat surface to form a 1.5 cm thick layer, and then sterilized under ultraviolet light for 2 h, and then stored in sand at 19℃ for 78 days to obtain Epimedium brevicornum seeds with morphologically after-ripened embryos; wherein the volume ratio of the seeds to the sand is 4:1. The sand contains water in an amount of 11% by mass, and the sand is sterilized at 120℃ for 40 min.

[0064] S5. Layering treatment: the Epimedium brevicornum seeds with morphologically after-ripened embryos obtained in S4 are screened to remove rotten and deteriorated seeds, and then sterilized under ultraviolet light for 2 h, and then subjected to 12 mg / L flumetralin seed soaking treatment for 6 h and 0.05 mg / L thiabendazole seed soaking treatment for 6 h, respectively, and then mixed with a sand storage medium in a bean curd transfer basket 645x445x195mm with a wall thickness of more than 2 mm with rock wool laid at the bottom, and then stored at 5.0℃ for 120 days, so that the ABA concentration of the endosperm and seed coat is lower than 1 mg / L, and the gibberellin content of the whole seed is more than 2 mg / L, and then the layering treatment is completed. The sand storage medium is a mixture of sand and sawdust, and the volume ratio of the seed, sand and sawdust is 1:3:2. The sand storage medium contains water in an amount of 12% by mass, and the sand is sterilized at 120℃ for 40 min.

[0065] S6. Sampling and checking the embryo development status, and when 90% of the seeds reach the cotyledon stage, the seeds can be sown.

[0066] An Epimedium brevicornum breeding method, and the specific steps are as follows:

[0067] 1). Disinfection: the obtained Epimedium brevicornum seeds after ripening are subjected to disinfection, and the specific steps are as follows: first, the seeds are treated with 75% ethanol for 60 s, then the seeds are treated with 1% hypochlorite solution diluted 8 times for 12 min, and finally the seeds are washed with sterile water for 5 times, so that the seeds after disinfection are obtained.

[0068] 2). The seeds after disinfection are inoculated into a culture bottle containing culture medium for 35 days, so that Epimedium brevicornum seedlings are obtained; the culture medium comprises WPM culture medium+AC 0.5 g / L+vermiculite 30 g / L+cane sugar 30 g / L+IBA 0.5+IAA 0.4+GA 0.8, and the pH value is 6.0.

[0069] 3). After the root length of the Epimedium brevicornum seedlings in 2) is 2 cm, and 3 true leaves grow out, the culture bottle cap is gradually opened for seedling acclimation for one week, and then the Epimedium brevicornum seedlings are transplanted into a seedling raising box containing a special Epimedium brevicornum seedling raising medium. The medium is prepared according to the volume ratio of grass carbon soil: 2 mm particle size vermiculite: 3 mm particle size perlite = 3:5:2, and 5 L of a chlorine dioxide solution with a content of 40 mg / L is sprayed to wet 0.1 m 3 of the medium, and then the medium is sealed overnight.

[0070] The base of the seedling box is a 72-hole seedling plug, which is filled with the substrate of item 3. When transplanting, the seedlings are first washed with 25°C clean water, then disinfected with 0.2% potassium permanganate for 40 minutes, and then washed again with clean water. A small shallow pit is made in the center of the substrate of the plug with a 0.5 cm wide bamboo sheet, and the seedling is planted in the pit. The base of the seedling is lightly pressed with the index finger and middle finger, and then water is sprayed. Finally, a seedling box cover with air windows is put on, and the two air windows on the cover are opened.

[0071] Comparative Example 1

[0072] The Epimedium seed is collected, and then air-dried for 50 hours in a room with an air humidity of 40-50%. The fruit is observed to be cracked to more than 65%. The seed is disinfected with an extract of Sophora flavescens, an extract of Cnidium monnieri, and an extract of Phellodendri chinensis. The extract is prepared as follows: 500 g of Sophora flavescens, 500 g of Cnidium monnieri, and 500 g of Phellodendri chinensis are added to 6 L of deionized water, and then ultrasonic-assisted extraction is performed at 48°C for 36 minutes at a power of 500 W. The mixture is centrifuged at 2000 rpm for 3 minutes, and the filtrate is collected to obtain the extract. The seed is disinfected for 0.5 hours, and then soaked in 50 mg / L gibberellin for 3 hours. After being washed with sterile water, the seed is sown in a seedling tray containing vermiculite:sand=3:2. After 35 days, the germination rate is 0%.

[0073] Comparative Example 2

[0074] The Epimedium seed is collected, and then disinfected with an extract of Sophora flavescens, an extract of Cnidium monnieri, and an extract of Phellodendri chinensis for 0.5 hours. The seed is sprayed with 5 mg / L gibberellin, and then stored in a food preservation box at a temperature of 2°C for 7 days. The seed is disinfected with 70% alcohol, washed with sterile water for 3 times, disinfected in a 5% sodium hypochlorite solution for 6 minutes, and then washed with sterile water for 5 times. The seed is peeled on a super-clean bench, and then sown in a culture medium WPM medium+AC 0.5 g / L+vermiculite 30 g / L+cane sugar 30 g / L+IBA 0.5+IAA 0.4+GA 0.8, pH=6.0. After 35 days, the germination rate is 12.79%. The extract is prepared as follows: 500 g of Sophora flavescens, 500 g of Cnidium monnieri, and 500 g of Phellodendri chinensis are added to 6 L of deionized water, and then ultrasonic-assisted extraction is performed at 48°C for 36 minutes at a power of 500 W. The mixture is centrifuged at 2000 rpm for 3 minutes, and the filtrate is collected to obtain the extract.

[0075] After 35 days, the germination rates of the seedling trays in Examples 1-3 and Comparative Examples 1-2 are counted, as shown in Table 1.

[0076] Table 1: Germination rates of seedling trays in different examples and comparative examples

[0077] Germination rate (%) Example 1 85.93 Example 2 86.00 Example 3 86.72 Comparative Example 1 0 Comparative Example 2 12.79

[0078] From Table 1, it can be seen that by using the seed embryo morphological after-ripening liquid, temperature variation combined with growth regulating substances, physiological after-ripening is synchronized, and the seed embryo rate reaches 93.19%. The after-treated Epimedium seed is planted in the after-ripening medium and the seedling raising medium, and the seedling raising has a germination rate of up to 86.72%, which is much higher than that of the comparative example 1 and the comparative example 2.

[0079] While the preferred embodiments of the application have been described, modifications and variations can be apparent to those skilled in the art once aware of the general underlying concepts. Accordingly, the appended claims intend to include within their scope all such modifications and variations as fall within the scope of the application.

[0080] Obviously, various modifications and changes can be made to the present application by those skilled in the art without departing from the spirit and scope of the application. Accordingly, it is intended that the present application embrace all such modifications and changes as fall within the scope of the appended claims and their equivalents.

Claims

1. A method for post-ripening of Epimedium seeds, characterized in that, Specifically, the following steps are included: Seed embryo morphology after-ripening solution treatment: Epimedium seeds were soaked in 300-400 mg / L ethephon for 8-12 hours and then rinsed clean; they were then transferred to a physiological after-ripening solution and soaked for 3-4 days to obtain Epimedium seeds with physiological after-ripening catalyzed by soaking; the physiological after-ripening solution was a nitrogen, phosphorus, potassium, calcium, boron, and zinc seed embryo morphology after-ripening solution, which contained nitrogen 0.6-0.9 g / L, phosphorus 0.1-0.3 g / L, potassium 0.5-0.8 g / L, calcium 0.05-0.2 g / L, boron 2-5 mg / L, and zinc 1.5-4.0 mg / L; Morphological after-ripening storage: After soaking, the Epimedium seeds, which have undergone physiological after-ripening, are disinfected for 1-2 hours, and then stored in sand at 15-19℃ for 58-78 days to obtain Epimedium seeds that have completed embryo morphological after-ripening; wherein, the volume ratio of seeds to sand is 1:

4. Stratification treatment: Epimedium seeds that have completed embryo morphology and post-ripening are treated with 4-12 mg / L fluazinam for 4-6 h and 0.02-0.05 mg / L thiafenuron for 4-6 h respectively, and then stored in a sandy substrate at 2-5℃ for 90-120 days. The sandy substrate is a mixture of sand and sawdust in a volume ratio of:seed:sand:sawdust = 1:3:

2.

2. A method for breeding Epimedium, characterized in that, The specific steps for propagating Epimedium using Epimedium seeds obtained through the post-ripening method of Epimedium seeds as described in claim 1 are as follows: Epimedium seeds obtained by the after-ripening method were buried in a culture medium for 30-35 days to obtain Epimedium seedlings; the culture medium consisted of WPM medium + AC 0.5 g / L + vermiculite 30 g / L + SUGAR 30 g / L + IBA 0.5 mg / L + IAA 0.4 mg / L + GA 0.8 mg / L, pH=6.0; Epimedium seedlings were transplanted into a seedling substrate for propagation; the seedling substrate was prepared by volume ratio of peat moss: vermiculite: perlite = 3:5:

2.

3. The method for breeding Epimedium according to claim 2, characterized in that, The seedling substrate is placed in the seedling box, the base of the seedling box is a 72-cell seedling tray, and the lid of the seedling box has two air windows.

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