Method for producing Gastrodia elata seed using Armillaria mellea using dual-purpose fungus

Abstract

The present invention provides a method for producing Gastrodia elata seed hemp by using Armillaria mellea, comprising: (1) adding a culture medium of Armillaria mellea to a Hubei crabapple extract, and then adding Gastrodia elata seeds and soaking them in the culture medium to obtain pretreated seeds; (2) sowing the pretreated seeds on a solid culture medium, storing them in the dark at 3-10°C, and then culturing them in the dark at room temperature, thereby achieving the dual-use of Armillaria mellea and facility-based production of Gastrodia elata seed hemp. In the technical solution of the present invention, Armillaria mellea contains multiple enzymes such as xylanase and laccase, which can destroy the seed coat of Gastrodia elata seeds, allowing water, nutrients, and germination-promoting substances (such as phlorizin) to enter the Gastrodia elata seeds. The dual-use of Armillaria mellea is achieved, that is, during the germination stage of Gastrodia elata seeds, the multiple enzymes produced by Armillaria mellea can degrade lignin in the seed coat, allowing germination-promoting substances and nutrients to enter the seeds, thereby providing nutrition to Gastrodia elata during its growth period.

Description

Technical Field

[0001] The invention belongs to the field of Gastrodia elata planting agricultural technology, and relates to Gastrodia elata seed germination and a Gastrodia elata planting facility production method. Background Art

[0002] Gastrodia elata ( Gastrodia elata Gastrodia elata (Bl.) is a commonly used and valuable traditional Chinese medicine in my country. It is a perennial, heterotrophic herbaceous orchid that lives in symbiosis with fungi. Its rhizomes are used as medicine. Its properties are sweet, slightly pungent, warming, non-toxic, and liver-regulating. It has the effects of invigorating qi, calming the liver, dispelling wind, relieving asthma, and promoting blood circulation. Modern research has revealed that Gastrodia elata possesses multiple pharmacological effects, including anticonvulsant, hypnotic, sedative, analgesic, anti-epileptic, anti-inflammatory, intellectual, immunomodulatory, and anti-aging properties. It is used to treat a variety of conditions, including hypertension, headaches, vertigo, rheumatism, and low back and leg pain.

[0003] Since Gastrodia elata seeds have no leaves, roots, or endosperm, and cannot absorb nutrients from the environment on their own, the germination of Gastrodia elata seeds requires the genus Trichoderma of the family Trichoderma ( Mycena ) fungi, such as Dendrobium officinale ( Mycena dendrobii ) and Osmanthus fragrans ( M.osmundicola Lange ) provide nutrients for germination of protocorms, which then differentiate into vegetative propagation stems. However, the growth and development of Gastrodia elata vegetative propagation stems cannot be satisfied by digestion of germination bacteria alone, and must be combined with Armillaria mellea ( Armillariella mellea Normal growth is achieved through the establishment of a nutritional relationship between Gastrodia elata and Armillaria mellea, which is characterized by mutual dependence and mutual benefit.

[0004] Orchid seeds typically germinate through two methods: symbiotic germination and non-symbiotic germination. Symbiotic germination occurs when orchid seeds germinate in a specific substrate through the infection of fungi. Because orchid seeds lack endosperm, nutrients are insufficient for natural germination. Some orchid seeds lack various enzymes and are unable to utilize their own nutrients. Certain fungi can secrete plant hormones or produce extracellular enzymes to aid in germination. Therefore, symbiotic germination technology has been applied to the germination of many orchid seeds. Thirteen major germination fungi are found in Gastrodia elata, 12 of which belong to the genus Agaricus in the class Basidiomycetes. Agaricus odoratus and Agaricus dendrobium have been the most researched and applied species.

[0005] Non-symbiotic germination refers to the technique of germinating orchid seeds on artificial culture media without the need for infection by the target fungus. Conventional non-symbiotic germination methods include the selection of basal culture media, phytohormones, organic additives, and certain bacterial extracts, as well as the adjustment of temperature, light intensity, and culture pH. Commonly used basal media include MS, modified MS, RE, Nistch, and N6, though the optimal medium varies depending on the species. Regarding phytohormones, orchid seeds generally germinate in germination media without the addition of phytohormones. However, some species can increase germination rates, speed up germination, and accelerate growth with the addition of low concentrations of phytohormones (such as 6-BA, NAA, or 2,4-D). Organic additives commonly used in tissue culture, such as coconut water, banana juice, and potato juice (purée), effectively promote seed germination. However, the effectiveness of these additives varies depending on the orchid species, and inappropriate organic additives can actually inhibit germination. Furthermore, some bacterial extracts can also affect orchid seed germination. For example, ergosterol extracted from the ethanol extract of Armillaria cordi can significantly increase the germination rate of Gastrodia elata seeds. Regarding temperature, it's generally believed that the optimal germination temperature for orchid seeds is around 25°C, but different orchid species have their own optimal temperature ranges. The same is true for light: some orchids require full darkness or full light, while others require a semi-dark and semi-light environment. For example, Gastrodia elata seeds don't require light to germinate, while the germination rate of Bletilla striata seeds cultured in the dark is significantly lower than those cultured in the light.

[0006] Research on non-symbiotic germination of Gastrodia elata is limited, primarily focusing on the 1980s. Gastrodia elata seeds can germinate on both 1 / 2 MS medium and Vacin and Went medium. However, non-symbiotic germination of Gastrodia elata has lower germination rates, slower growth, and poorer resistance than symbiotic germination, significantly failing to meet practical production requirements. Currently, no non-symbiotic germination methods have been applied in practical production.

[0007] Both symbiotic and asymbiotic germination methods present disadvantages, severely impacting the development of the Gastrodia elata industry. Therefore, this study integrates the advantages of both symbiotic and asymbiotic germination methods. By adding exogenous germination-promoting substances and breaking seed dormancy at low temperatures, Armillaria mellea overcomes seed coat barriers and provides nutrients for Gastrodia elata growth. This innovation represents a key technology for seed germination in Gastrodia elata production, achieving dual-purpose use of Armillaria mellea, and is now being applied to the production of Gastrodia elata seeds in facilities. Summary of the Invention

[0008] The method of producing Gastrodia elata seed hemp by using Armillaria mellea as a single fungus and dual purpose is as follows: (1) using enzymes produced by Armillaria mellea culture to degrade lignin in the seed coat of Gastrodia elata seeds. This allows germination-promoting substances and nutrients to enter the interior of the Gastrodia elata seeds, providing conditions for seed germination; (2) treating at a low temperature of 3-10°C to overcome the inhibitory effect of Armillaria mellea on Gastrodia elata seed germination and break the dormancy of Gastrodia elata seeds; (3) using germination-promoting substances to promote Gastrodia elata seed germination; (4) using Armillaria mellea as a single fungus and dual purpose to produce Gastrodia elata seed hemp in a facility. The Armillaria mellea dual purpose of the present invention means that Armillaria mellea is used for both Gastrodia elata seed germination and Gastrodia elata growth. This changes the original use of two fungi in Gastrodia elata production: the germination fungi for Gastrodia elata seed germination and the Armillaria mellea providing nutrients for Gastrodia elata growth, to using only one fungi, Armillaria mellea. The main function of Armillaria mellea in the germination stage of Gastrodia elata seeds is to produce enzymes to destroy the seed coat of Gastrodia elata seeds and allow germination-promoting substances to enter the seeds.

[0009] The method for producing Gastrodia elata seed hemp comprises the following steps:

[0010] (1) Adding the honey fungus culture solution to the Malus hupehensis extract, and then adding the Gastrodia elata seeds and soaking them in the culture solution to obtain pretreated seeds;

[0011] (2) The pretreated seeds are sown on a solid culture medium, stored in the dark at 3-10°C, and then cultured in the dark at room temperature to break the dormancy of the Gastrodia elata seeds, thereby achieving the dual purpose of Armillaria mellea and the facility-based production of Gastrodia elata seeds.

[0012] The dark culture process can be performed in an opaque container or a container-shaded culture, a canopy-shaded culture, or a box-covered culture. During the dark culture process, air permeability and a high water content are maintained, and the air humidity environment is adjusted according to the season, such as about 20-80%.

[0013] In some embodiments, the Gastrodia elata seeds pretreated in step (1) are added to the culture medium, and the Armillaria mellea fungus solution is added, and the culture medium is sealed in a bag, stored in a dark place at low temperature, and then cultured in a dark place at room temperature, so as to realize the dual use of Armillaria mellea fungus and produce Gastrodia elata seeds in a facility-based manner.

[0014] The culture medium for culturing Armillaria mellea described in the present invention is a nutrient solution commonly used in the art and can be purchased commercially or homemade. The homemade Armillaria mellea culture medium is a PDA culture medium. The Armillaria mellea culture method comprises removing the strain from a preserved slant tube of Armillaria mellea, activating it on a PDA culture plate for 5 days, and then quantitatively inoculating the Armillaria mellea into the liquid culture medium using a hole punch. The culture is then cultured in a shaker at 25°C and 120 rpm for 3 to 5 days. The bacterial solution is filtered through a 0.22 μm microporous filter membrane to obtain a culture medium for use. The obtained culture medium of Armillaria mellea has a laccase activity of not less than 1000 U / L and a xylanase activity of not less than 50 U / L.

[0015] The Gastrodia elata seeds described herein are obtained from mature, unopened capsules. A specific seed extraction method involves placing the capsule in a nylon mesh bag and gently rinsing it with running water for 5 minutes. The capsule is then disinfected in a laminar flow hood with 75% alcohol for 30 seconds, rinsed three times with sterile water for 60 seconds each, then placed in a 0.1% mercuric chloride solution for 10 minutes, and rinsed four to five times with sterile water for 60 seconds each. The sterilized capsule is gently wiped dry with sterilized filter paper, carefully peeled open with sterile tweezers, and the Gastrodia elata seeds are shaken off. The capsule is then stored in a sterilized EP tube.

[0016] The Hubei crabapple extract is obtained by refluxing the crabapple in a 70-95% ethanol solution. It mainly contains flavonoids, mainly phlorizin, with a phlorizin content greater than 20%. The phlorizin content is measured before use, and the dosage is calculated based on phlorizin.

[0017] The amount of the Malus hupehensis extract or phlorizin is 0.01-50 mg / L, i.e., any value within the range of 0.01-50 mg / L. In some embodiments, the amount of the Malus hupehensis extract or phlorizin is 0.1-10 mg / L, i.e., any value within the range of 0.1-10 mg / L, such as 0.1 mg / L, 1 mg / L, or 10 mg / L in some embodiments.

[0018] In step (1), the Gastrodia elata seeds are soaked in the culture medium for 24-72 hours, such as 24 hours, 48 ​​hours, or 72 hours in some embodiments.

[0019] The solid culture medium in step (2) of the present invention is a modified PDA, which is also a technical effect that can be achieved by solid culture media well known in the art.

[0020] Solid culture medium (modified PDA) composition: 200 g potatoes, 20 g glucose, 4 g peptone, 50 g wheat bran, 1.5 g potassium dihydrogen phosphate, 1 g magnesium sulfate, 10 mg vitamin B1, 15-20 g agar, 1000 mL water, natural pH.

[0021] Preparation method: Wash and peel the potatoes, cut into small pieces, add water and boil (boil for 20-30 minutes), filter, add water and boil the wheat bran, filter, combine the filtrate, heat, add 15-20g agar, continue heating and stirring to mix, after the agar is dissolved, add glucose, peptone, potassium dihydrogen phosphate, magnesium sulfate, and vitamin B1 and stir evenly, cool slightly and then add water to 1000ml, package, sterilize (115℃) for 30 minutes, take out and cool for later use.

[0022] The liquid culture medium (i.e., the culture fluid) of the present invention is also a commonly used culture medium in the art. No agar is added, and the other components are the same as those of the solid culture medium (modified PDA) described above. The volume is adjusted to 1000 ml.

[0023] The low-temperature dark storage in step (2) is dark storage at 3-10°C for 7-10 days; the room temperature dark culture time is 5-10 weeks.

[0024] The culture medium in step (3) of the present invention can be a solid culture medium or a culture medium obtained by mixing sawdust, bran, corn, and gypsum and adding water to obtain a culture medium with a water content of 70-80%, which is obtained by bagging and sterilizing; the mass fractions of sawdust, bran, corn, and gypsum are 60-80%, 15-25%, 8-15%, and 1-2%. In conventional use, it is often necessary to bag the culture medium, such as by placing it in a polyethylene bag and then sterilizing it under high pressure. In conventional experiments, the bagging is often kept with a certain porosity to prevent the culture medium from boiling and splashing during high-pressure sterilization. High-pressure sterilization is often maintained at 121°C for 60 minutes.

[0025] The culture medium can also be nutrient soil, which also needs to be disinfected before use. The nutrient soil was purchased from Pinshitop Horticulture (Shanghai) Co., Ltd. After adding seeds to the nutrient soil, it was spread flat in a nylon mesh bag under a sterile environment, the bag was sealed, and the nylon mesh bag was covered with 3 to 4 cm of sawdust. After adding honey fungus, it was covered with nutrient soil. As long as it is possible to add seeds to the culture medium or nutrient soil, and then add honey fungus for cultivation, the dual-purpose honey fungus described in this application can be achieved, and Gastrodia elata seed hemp can be produced in a facility-based manner.

[0026] The solid culture medium can also be culture soil formed by sterilizing sand, leaves, sawdust, or humus.

[0027] The low-temperature dark storage in step (3) is dark storage at 3-10°C for 7-10 days; the room temperature dark culture time is 5-10 weeks.

[0028] In the technical solution of the present invention, Armillaria mellea contains multiple enzymes, such as xylanase and laccase, that can break down the seed coat of Gastrodia elata seeds, allowing water, nutrients, and germination-promoting substances (such as phlorizin) to enter the seeds. Armillaria mellea does not produce germination-promoting substances and cannot promote germination of Gastrodia elata seeds. In addition, it produces substances that inhibit germination, inhibiting germination. Pre-treatment and storage at low temperatures help overcome this inhibitory effect and break the dormancy of Gastrodia elata seeds. After germination, Gastrodia elata seeds can directly interact with Armillaria mellea and begin growth. Simulated field germination culture results show that Gastrodia elata seeds begin to grow after germination, and their morphology is significantly larger than that of germination cultures grown in culture medium. The results indicate that combining Malus chinensis extract or phlorizin with Armillaria mellea can promote germination and growth of Gastrodia elata seeds. This achieves a dual purpose for Armillaria mellea: during the germination stage of Gastrodia elata seeds, the multiple enzymes produced by Armillaria mellea can degrade lignin in the seed coat, allowing germination-promoting substances and nutrients to enter the seeds, providing nutrition for Gastrodia elata during its growth period. The germination of Gastrodia elata seeds does not require germination fungi, which achieves a breakthrough in the production model of Gastrodia elata, reducing the number of Gastrodia elata production from two fungi (germination fungi and Armillaria mellea) to one fungi (Armillaria mellea). BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 These are morphological diagrams of Gastrodia elata seeds at various stages of germination under a microscope.

[0030] Figure 2 This is the morphological diagram of Gastrodia elata seeds germination cultured in phlorizin-promoting culture medium.

[0031] Figure 3 The germination morphology of Gastrodia elata seeds cultured to simulate field germination promoted by phlorizin.

[0032] Figure 4 Plant Gastrodia elata in boxes.

[0033] Figure 5 To plant Gastrodia elata in bags. DETAILED DESCRIPTION

[0034] Example 1

[0035] The mature, unopened Gastrodia elata capsules, Armillaria mellea, and sawdust used in this study were all provided by Wufeng Fenglin Fungus Planting Co., Ltd. Hubei Begonia extract and phlorizin (purity ≥ 98%, extracted from Hubei Begonia leaves) were provided by the Hubei Key Laboratory of Natural Product Research and Utilization (Three Gorges University). Nutrient soil was purchased from Pinshitop Horticulture (Shanghai) Co., Ltd. Nylon mesh bags with a pore size of 20 μm and a size of 3 × 3 cm were purchased from Lifei Screen Factory.

[0036] Solid medium (modified PDA): 200g potatoes, 20g glucose, 4g peptone, 50g wheat bran, 1.5g potassium dihydrogen phosphate, 1g magnesium sulfate, 10mg vitamin B1, 15-20g agar, 1000mL water, natural pH. Preparation: Wash, peel, and cut the potatoes into small pieces. Boil in water (20-30 minutes), filter. Boil the wheat bran, filter, and combine the filtrates. Heat the mixture, then add 15-20g agar. Continue heating and stirring to mix thoroughly. Once the agar has dissolved, add the glucose, peptone, potassium dihydrogen phosphate, magnesium sulfate, and vitamin B1, and stir thoroughly. Allow to cool slightly, then add water to 1000mL. Aliquot and sterilize at 115°C for 30 minutes. Remove and cool until ready for use.

[0037] Liquid medium: Omit the agar. The other ingredients are the same as those for the solid medium (modified PDA). Bring the volume to 1000 ml.

[0038] Hubei Begonia Extract: Hubei Begonia ( Malus hupehensis The product obtained by refluxing the leaves of Pamp. Rehd. with 85% ethanol contains flavonoids, primarily phlorizin. The phlorizin content should be measured before use, and the dosage should be calculated based on the phlorizin content.

[0039] Experiment on germination of Gastrodia elata seeds

[0040] (1) Capsule disinfection: Place the mature, unopened capsules in a nylon mesh bag and rinse gently with running water for 5 minutes. Then, disinfect them in a clean bench with 75% alcohol for 30 seconds, rinse with sterile water three times for 60 seconds each, then disinfect them in 0.1% mercuric chloride solution for 10 minutes, and rinse with sterile water 4-5 times for 60 seconds each. After disinfection, gently wipe the capsules dry with sterilized filter paper, carefully peel them open with sterile tweezers, and shake off the Gastrodia elata seeds. Store them in a sterilized EP tube.

[0041] (2) Stereomicroscope observation: Take the culture medium mixed with Gastrodia elata seeds after 4-8 weeks of culture and apply it on a glass slide. The morphological characteristics of Gastrodia elata seeds at various germination stages are observed under a stereomicroscope. Observation is conducted every 10 days.

[0042] (3) Culture of Armillaria mellea

[0043] After being activated for 5 days on a PDA culture medium plate, the Armillaria mellea was taken out from the preserved slant test tube of Armillaria mellea and quantitatively inoculated into the liquid culture medium using a hole puncher. The culture was cultured in a shaker at 25°C and 120 rpm for 3 to 5 days. The bacterial liquid was filtered with a 0.22 μm microporous filter membrane to obtain the Armillaria mellea culture medium for use.

[0044] (4) Gastrodia elata seed germination treatment

[0045] Germination culture: Phlorizin or Malus hupehensis extract was added to Armillaria culture medium (final concentrations of phlorizin or Malus hupehensis extract were 0.01, 0.1, 1, 10, and 50 mg / L, respectively. In some experiments, filtration was performed using a 0.22 μm microporous membrane to remove impurities). Sterilized Gastrodia elata seeds were pretreated by soaking in the culture medium for 48 hours to obtain pretreated Gastrodia elata seeds. A blank control group of Gastrodia elata seeds pretreated with Armillaria culture medium for 48 hours was used.

[0046] The treated Gastrodia elata seeds were evenly spread on a solid culture medium and stored in the dark at 4°C for 8 days to break the dormancy of the Gastrodia elata seeds. They were then transferred to a dark culture at room temperature of 20-25°C for 8 weeks.

[0047] To simulate field germination, sterilize the nutrient soil and place it in a culture flask. Aseptically spread the sterilized and pretreated Gastrodia elata seeds (germinated using the same medium) into a 3x3 cm nylon mesh bag with a 20 μm pore size and seal the bag tightly. Cover the nylon mesh bag with 3-4 cm of sawdust. Add 5 mL of Armillaria mellea fungus solution using a pipette and cover with 1-2 cm of nutrient soil. Store in the dark at 4°C for 7-10 days, then transfer to room temperature (20-25°C) and incubate in the dark for 8 weeks.

[0048] After 8 weeks, the germination of Gastrodia elata seeds in each group was observed under a stereomicroscope, and five fields of view of each sample were taken and photographed for analysis.

[0049] The data were analyzed for variance and significant difference using SPSS 22.0 software.

[0050] Gastrodia elata seed germination results

[0051] (1) Morphological observation results: Figure 1 As shown, mature Gastrodia elata seeds are long and narrow spindle-shaped; one end is blunt and the other end is pointed; the seed coat is translucent and the embryo is light yellow. From the outside to the inside of the seed are the seed coat, air cavity and proembryo. After observing and sampling Gastrodia elata seeds at different stages under a stereo microscope at different time periods, it was found that the changes in the Gastrodia elata embryo during the germination process are divided into the following six stages. The embryo has not changed (stage 1), the embryo swells but has not yet broken through the seed coat (stage 2), the embryo swells and breaks through the seed coat (stage 3), the embryo has meristematic primordium (stage 4), the bud is formed (stage 5), and the bud elongates (stage 6). The germination rate calculation formula is as follows:

[0052]

[0053] (2) Effects of different concentrations of phlorizin and Malus hupehensis extract on Gastrodia elata seed germination

[0054] Compared with the blank control, phlorizin or Malus hupehensis extract at concentrations of 0.01 mg / L, 0.1 mg / L, 1 mg / L, 10 mg / L, and 50 mg / L promoted germination of Gastrodia elata seeds (P < 0.05). The promoting effect of phlorizin or Malus hupehensis extract at a concentration of 0.1 mg / L was even more significant (P < 0.05), with Malus hupehensis extract exhibiting superior germination activity compared to phlorizin at the same concentration. The highest germination rate with phlorizin was 67.7%, 13 times that of the blank control, while the highest germination rate with Malus hupehensis extract was 73.52%, 14 times that of the blank control. Phlorizin is the primary germination-promoting compound in Malus hupehensis extract, and the optimal concentration of phlorizin is 0.1–1 mg / L. This is shown in Table 1.

[0055] Table 1 Effects of phlorizin and Malus hupehensis extract on the germination rate of seeds cultured in culture medium

[0056]

[0057] Among them, *P<0.05.

[0058] Morphological observations revealed that the changes in the Gastrodia elata embryos during germination in the blank control group were mainly stage 1, i.e., the embryos did not change, and a small amount could enter stage 2, i.e., the embryos swelled but had not yet broken through the seed coat. After phlorizin treatment, the changes in the Gastrodia elata embryos during germination were mainly stage 4 (appearance of meristematic primordium in the embryos) and stage 5 (bud formation), some were stage 3 (embryos swelled and broke through the seed coat) and stage 6 (bud elongation), and a small amount was stage 2. The changes in the Gastrodia elata embryos during germination after treatment with the Hubei crabapple extract were consistent with those of the phlorizin treatment. Figure 2 shown.

[0059] In the use of the culture medium of the present invention for germination culture, pretreatment for 24, 48, and 72 hours can promote the germination of Gastrodia elata seeds. There is no significant difference in the promoting effect between 48 and 72 hours, but it is higher than 24 hours.

[0060] The results of simulated field germination incubation were consistent with those of culture medium germination incubation. The results showed that phlorizin or Malus hupehensis extract at concentrations of 0.01 mg / L, 0.1 mg / L, 1 mg / L, 10 mg / L, and 50 mg / L promoted germination of Gastrodia elata seeds compared with the blank control (P < 0.05). The promoting effect of phlorizin or Malus hupehensis extract at a concentration of 0.1 mg / L was even more significant (P < 0.05), with Malus hupehensis extract exhibiting a superior promoting effect compared to phlorizin at the same concentration. Phlorizin is the primary germination-promoting compound in Malus hupehensis extract, and the optimal concentration for phlorizin is 0.1-1 mg / L. This is shown in Table 2.

[0061] Table 2 Effects of phlorizin and Malus hupehensis extract on the germination rate of seeds in simulated field germination culture

[0062]

[0063] Among them, *P<0.05.

[0064] Morphological observation showed that the changes of Gastrodia elata embryos during germination in the blank control group were mainly stage 1, and a small amount could enter stage 2. The changes of Gastrodia elata embryos during germination after phlorizin treatment were mainly stage 5 and stage 6, some were stage 3 and stage 5, and a small amount was stage 2. Figure 3 shown.

[0065] The changes in Gastrodia elata seed embryos during germination after treatment with Malus hupehensis extract were consistent with those observed with phlorizin. This indicates that phlorizin is the primary germination-promoting substance in Malus hupehensis. It promotes Gastrodia elata seed germination, with a germination rate exceeding 60%. Most seeds are already in the bud formation and elongation stages, indicating the potential for further growth. This has practical application value. Given the large number of Gastrodia elata seeds, the failure of some seeds to germinate does not affect production. Further research is warranted to improve germination rates.

[0066] Gastrodia elata seed germination facility seed production

[0067] Example 2

[0068] Box planting

[0069] The culture boxes, sand, leaves, humus, etc. used for box planting are all sterilized by ultraviolet irradiation or high-pressure steam sterilization (sterilization at 121℃ for 1h).

[0070] (1) Pretreatment of Gastrodia elata seeds: Add the extract of Malus hupehensis to the culture medium of Armillaria mellea (so that the final concentration of phlorizin is 0.1 mg / L). Soak the Gastrodia elata seeds in the culture medium for 48 hours.

[0071] (2) Box planting: The bottom of the cultivation box is covered with 3-4 cm thick sand, and a small amount of wet miscellaneous leaves are placed on it. The cultured Armillaria bags are placed 3-4 cm apart along the cultivation bed, and the intervals are about 10 cm. The Gastrodia seeds pre-treated in Example 1 are evenly sown in the gaps between the Armillaria bags (Example 1 was not found). The gaps between the sticks are filled with leaves or miscellaneous sawdust. After adding 2-3 cm of humus soil, a second layer is planted perpendicular to the lower layer. Plant 2-3 layers in total, and then cover the surface of the sticks with humus soil to a thickness of 3-4 cm. Cover with a lid and pay attention to ventilation. Store at 4-10℃ for 7-10 days, and then transfer to room temperature 20-25℃ for cultivation.

[0072] (3) Water control: When cultivating Gastrodia elata indoors, the honey fungus grows vigorously in spring, so the water content should be controlled at around 20%. From June to July, Gastrodia elata grows vigorously and the water requirement gradually increases. The water content should be controlled at around 50%. If the air humidity is high and evaporation is low, water lightly and ventilate the soil. Excessive watering is not conducive to the growth of Gastrodia elata.

[0073] (4) Ventilation: The surface of the plant tends to become hardened after watering, so loosen the soil in time to facilitate ventilation.

[0074] Figure 4 These are the attached pictures of Gastrodia elata seeds planted in boxes, where A is Gastrodia elata planted in different foam plastic boxes, and B is the picture of Gastrodia elata seeds obtained after maturity.

[0075] Harvesting

[0076] After 8-12 months of cultivation, the seed hemp is mature and is usually harvested in the spring before field planting. The hemp is unpacked or unpacked, the covering layer and culture medium removed, and the hemp (white rice) is sorted out. The hemp is sold in whole boxes or bags, and can be directly removed for planting. Figure 5 These are the attached pictures of Gastrodia elata cultivated in bags, where A is Gastrodia elata cultivated in different polyethylene bags, and B is the picture of Gastrodia elata obtained after maturity.

[0077] Example 3

[0078] Bag planting

[0079] (1) Culture of Armillaria in bags: The culture medium is 70% sawdust, 19% bran, 10% corn, 1% gypsum, and 70% water. Mix the above culture medium in proportion and add water to adjust the moisture content to the corresponding ratio. After the culture medium is fully mixed, it is placed in a polyethylene corner bag. The height of each bag should not exceed 2 / 3 of the total height of the bag to prevent the culture medium from boiling and splashing during high-pressure sterilization. High-pressure sterilization is performed at a temperature of 121°C for 60 minutes. After the sterilization is completed, the pressure is naturally cooled and reduced. Conventional inoculation of Armillaria liquid culture is carried out and cultured at 20-25°C.

[0080] (2) Seed Mixing and Cultivation: Take the freshly prepared and disinfected Armillaria mellea bag material, add 1 / 3 to 1 / 2 of the grown Armillaria mellea bag material, mix well, then add the Gastrodia elata seeds pretreated in Example 1, mix well, bag naturally (without squeezing), and seal. Store at 4-10°C for 7-10 days, then transfer to room temperature (20-25°C) for cultivation. Ensure ventilation and moisture retention in the cultivation room, with an air humidity of about 50-80%.

[0081] Harvesting is the same as in Example 2.

Claims

1. A method for producing Gastrodia elata seeds using Armillaria mellea as a dual-purpose fungus, characterized in that: The steps include: (1) Adding a culture solution of Armillaria mellea to a Hubei crabapple extract, and then adding Gastrodia elata seeds and soaking them in the culture solution to obtain pretreated seeds, wherein the Hubei crabapple extract is a product obtained by refluxing the leaves of the Hubei crabapple with a 70-95% ethanol solution, and the amount of the Hubei crabapple extract is 0.01-50 mg / L; (2) The pretreated seeds are sown on a solid culture medium, stored in the dark at 3-10°C, and then cultured in the dark at room temperature, so as to achieve dual-purpose use of Armillaria mellea and to produce Gastrodia elata seeds in a facility-based manner.

2. A method for producing Gastrodia elata seeds using Armillaria mellea as a dual-purpose fungus, characterized in that: The steps include: (1) adding phlorizin to the culture solution of Armillaria mellea, and then adding Gastrodia elata seeds and soaking them in the culture solution to obtain pretreated seeds, wherein the amount of phlorizin used is 0.01-50 mg / L; (2) The pretreated seeds are sown on a solid culture medium, stored in the dark at 3-10°C, and then cultured in the dark at room temperature, so as to achieve dual-purpose use of Armillaria mellea and to produce Gastrodia elata seeds in a facility-based manner.

3. The method for producing Gastrodia elata seeds by using Armillaria mellea as a dual-purpose fungus according to claim 1 or 2, wherein: In step (1), the Gastrodia elata seeds are soaked in the culture medium of Armillaria mellea for 24-72 hours.

4. The method for producing Gastrodia elata seeds by using Armillaria mellea for dual purposes according to claim 1 or 2, wherein: The laccase activity in the culture medium is not less than 1000U / L; the xylanase activity is not less than 50U / L.

5. The method for producing Gastrodia elata seeds by using Armillaria mellea for dual purposes according to claim 1 or 2, characterized in that: In step (2), the low-temperature dark storage is dark storage at 3-10°C for 7-10 days; the room temperature dark culture time is 5-10 weeks.

6. The method for producing Gastrodia elata seeds by using Armillaria mellea as a dual-purpose fungus according to claim 1 or 2, characterized in that: The solid culture medium described in step (2) is a modified PDA culture medium or nutrient soil.

7. The method for producing Gastrodia elata seeds by using Armillaria mellea for dual purposes according to claim 1 or 2, characterized in that: The solid culture medium described in step (2) is a culture medium with a water content of 70-80% obtained by mixing sawdust, bran, corn, and gypsum and then adding water, and the culture medium is bagged and sterilized.

8. The method for producing Gastrodia elata seeds by using Armillaria mellea for dual purposes according to claim 1 or 2, characterized in that: The solid culture medium described in step (2) is a culture soil formed by sterilizing sand, leaves, sawdust, and humus.

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