Method for promoting seed germination of cremastra appendiculata

Abstract

The invention provides a method for promoting seed germination of cremastra appendiculata, which comprises the following steps: selecting mature cremastra appendiculata capsules, and taking out seeds in the cremastra appendiculata capsules; uniformly mixing the seeds in the five rhododendron capsules with one crushed grain coprinus comatus cultivated species; a mixture of the seeds and the grain coprinus comatus cultivated species is put into a transparent adhesive bag, the humidity is controlled to be 60-80%, and a bag opening of the transparent adhesive bag is sealed; 2-5 air holes are evenly formed in each of the two sides of the transparent adhesive bag, and the hole diameter is 0.5-1.5 cm; and placing the transparent adhesive bag filled with the mixture in an environment with the illumination intensity of 0lx, the relative humidity outside the transparent adhesive bag of 60-80% and the temperature of 18-22 DEG C until protocorms are generated. According to the method, azalea seeds and crushed grain coprinus comatus cultivated species are evenly mixed according to the proportion and then put into a transparent plastic bag, full darkness, the constant temperature of 18-22 DEG C, the constant humidity environment of 60-80% and ventilation of the two sides of the plastic bag are controlled, the seed germination starting time and the time for forming protocorms with the diameter of 0.5 cm are short, operation is easy, cost is low, and the seed germination rate is high.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of planting, in particular to a method for promoting seed germination of Cymbidium goeringii. BACKGROUND

[0002] Seed germination of Cymbidium goeringii has important significance in orchid breeding and conservation. Cymbidiumspp Seed germination of Cymbidium goeringii is very difficult under natural conditions. Research on the causes of seed germination of Cymbidium goeringii found that factors leading to difficult seed germination of Cymbidium goeringii include incomplete embryo structure, seed coat inhibition, production of germination inhibitors, incomplete embryo development, mycorrhizal fungi, and external environment (temperature, light, pH value).

[0003] Due to the small size of Cymbidium goeringii seeds and the lack of endosperm and storage material, external carbon source and growth regulators are needed to support germination, and the germination rate is very low under natural conditions; it depends on symbiotic fungi to provide nutrition, but the symbiotic mechanism is complex and difficult to simulate artificially. Currently, artificial germination mainly studies some basic culture media, such as MS (Murashige & Skoog), KC (Knudson C), or 1 / 2MS medium, which commonly adds sucrose (20-30g / L) as a carbon source. Some plant growth regulators are added to the basic culture medium, such as cytokinin: 6-BA (0.5-2mg / L), auxin: NAA (0.1-0.5mg / L), etc., and some natural additives, such as coconut water (10-20%), banana mud or potato extract, are used to provide organic nutrients.

[0004] As can be seen from the above, the existing technology has the problems of high cost and great difficulty in artificial operation for promoting seed germination of Cymbidium goeringii, and therefore, there is an urgent need for a method with simple operation and high germination rate to solve the problems in the prior art. SUMMARY

[0005] The main purpose of the present application is to provide a method for promoting seed germination of Cymbidium goeringii, which at least solves the problems of high cost and great difficulty in artificial operation for promoting seed germination of Cymbidium goeringii in the prior art.

[0006] In order to achieve the above-mentioned purpose, the present application provides a method for promoting seed germination of Cymbidium goeringii, comprising: selecting mature Cymbidium goeringii capsules, and taking out the seeds in the Cymbidium goeringii capsules; mixing the seeds in 5 Cymbidium goeringii capsules with 1 part of fresh weight 450-550g of broken crystal button mushroom cultivation species; the crystal button mushroom cultivation species is a solid substrate whole containing active mycelium formed after sawdust, wheat bran and rice bran are mixed to prepare a culture medium, the culture medium is sterilized, inoculated with crystal button mushroom spores, and cultured until the mycelium completely covers the culture medium; The mixture of seeds and the cultivated species of Coprinus cinereus is put into a transparent plastic bag, and the humidity is controlled at 60-80%, and the bag opening is sealed. 2-5 evenly distributed air holes with a diameter of 0.5-1.5 cm are punched on both sides of the transparent plastic bag. The transparent plastic bag containing the mixture is placed in an environment with an illumination intensity of 0 lx, a relative humidity of 60-80% outside the transparent plastic bag, and a temperature of 18-22℃, until the protocorms are generated.

[0007] Optionally, the raw materials for the culture medium for preparing the cultivated species of Coprinus cinereus are mixed in a mass ratio of 7 parts of sawdust, 2 parts of wheat bran, and 1 part of rice chaff.

[0008] Optionally, a non-woven fabric patch is attached to the air hole.

[0009] Optionally, the non-woven fabric patch is a sterile medical-grade non-woven fabric with a grammage of 25-30 g / m 2 , a pore size of ≤0.1 mm, and an area that is 2-3 mm larger than the air hole diameter, completely covering the air hole and being fixed by a sterile adhesive tape.

[0010] Optionally, the particle size of the crushed cultivated species of Coprinus cinereus is 0.3-0.5 cm.

[0011] Optionally, before the seeds are mixed with the cultivated species of Coprinus cinereus, the seeds are wrapped with filter paper and then soaked in a 0.1%-0.3% sodium hypochlorite solution for 5-8 min, rinsed with sterile distilled water for 3-5 times, and the surface water is absorbed.

[0012] Optionally, the transparent plastic bag is a sterile polyethylene plastic bag with a thickness of 0.08-0.12 mm, a capacity of 50-100 mL, and the mixture of seeds and the cultivated species of Coprinus cinereus occupies 1 / 3-1 / 2 of the volume of the transparent plastic bag.

[0013] Optionally, in an environment with a temperature of 18-22℃, the transparent plastic bag is turned over once every 7-10 days: the bag is slowly turned over 180° from both sides, and the mixture in the bag is turned over synchronously, keeping it loose and uniform.

[0014] The present invention provides a method for promoting the germination of Rhododendron seeds, comprising: selecting mature Rhododendron capsules and extracting the seeds from the capsules; mixing the seeds from 5 Rhododendron capsules with 1 part of crushed Coprinus commune cultivar 450-550g fresh weight; placing the mixture of seeds and Coprinus commune cultivar into a transparent plastic bag, controlling the humidity at 60-80%, and sealing the bag opening; evenly punching 2-5 ventilation holes on each side of the transparent plastic bag, with a hole diameter of 0.5-1.5cm; placing the transparent plastic bag containing the mixture in an environment with a light intensity of 0lx, an external relative humidity of 60-80%, and a temperature of 18-22℃ until protocorms are generated. Therefore, by mixing azalea seeds with crushed crystalline Coprinus comatus cultivars in a certain proportion and then placing them in transparent plastic bags, only the environment of complete darkness, constant temperature of 18~22℃, constant humidity of 60~80% and ventilation on both sides of the plastic bag is required. The seed germination time and the time to form a protocorm with a diameter of 0.5cm are short. The operation is simple, low-cost and has a high seed germination rate. Attached Figure Description

[0015] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a flowchart of a method for promoting the germination of azalea seeds, which is an optional embodiment of the present invention. Detailed Implementation

[0016] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0017] like Figure 1 As shown, this application provides a method for promoting the germination of Rhododendron seeds, comprising: S1. Select mature rhododendron capsules and remove the seeds from the rhododendron capsules; S2. Mix the seeds from 5 rhododendron capsules with 1 part of 450-550g fresh-weight, crushed Coprinus coccinea cultivar; the cultivar is a solid matrix containing active mycelium formed by sterilizing a culture medium made of sawdust, wheat bran and rice husk, inoculating with Coprinus coccinea cultivar and culturing until the mycelium completely covers the culture medium. S3. Pack the mixture of seeds and crystalline Coprinus comatus cultivar into a transparent plastic bag, control the humidity at 60-80%, and seal the opening of the transparent plastic bag. S4. Make 2-5 ventilation holes evenly on each side of the transparent plastic bag, with a hole diameter of 0.5-1.5cm; S5, place the transparent plastic bag containing the mixture in an environment with an illumination intensity of 0 lx, a relative humidity of 60-80% outside the transparent plastic bag, and a temperature of 18-22℃ until the protocorms are generated.

[0018] Specifically, S1, select "mature Cymbidium ensifolium capsules", observe the appearance of the capsule, and generally mature capsules are yellowish brown, slightly hard in texture, without damage, insect damage or mold phenomenon. At this time, the embryo inside the seed has developed to the basic mature state, and the germination potential is stronger. If immature green capsules are selected, the seed embryo has not developed completely and is difficult to germinate, while over-mature capsules may cause the seeds to scatter or be contaminated by bacteria. When taking out the seeds, first clean the surface of the capsule (such as light flushing with sterile water), then use sterile scissors to cut open the capsule shell, and use sterile tweezers or a brush to gently brush the internal powdery seeds, avoiding forceful squeezing that may damage the seeds, and ensuring that the removed seeds remain dry and free of impurities. S2, mix all the seeds taken from 5 Cymbidium ensifolium capsules with 1 part of fresh weight 450-550g and broken treated grain spawn of Coprinus cinereus; 5 Cymbidium ensifolium capsules are mature capsules with different plant cross-pollination, growing period of 170-190 days, single fruit longitudinal diameter of 2-2.5 cm, and transverse diameter of 0.2-0.3 cm, ensuring that the seeds in the capsule develop completely and have good activity. The grain spawn of Coprinus cinereus is a solid substrate whole containing active mycelium, which is made of sawdust, wheat bran and rice chaff as raw materials, inoculated with Coprinus cinereus spores after sterilization, and cultured until the mycelium completely covers the culture medium. It can provide the necessary nutrients for the growth of Cymbidium ensifolium seeds. Since Cymbidium ensifolium seeds have no endosperm, they cannot independently obtain carbon, nitrogen and other nutrients, and must rely on the mycelium in the grain spawn to realize symbiotic nutrient supply. "5 capsule seeds with 450-550g fresh weight of grain spawn" can not only avoid excessive use of grain spawn leading to fungal competition for seed growth space, but also prevent insufficient nutrient allocation caused by excessive number of seeds, ensuring that each seed can obtain stable nutrients. The grain spawn is broken, not the raw material sawdust (although the sawdust itself is in the form of particles, but after combining with the mycelium to form the grain spawn, it will be in the form of blocks or agglomerates), but the whole solid substrate of the formed grain spawn is broken, so that it exists in the form of small particles. This treatment can significantly increase the contact area between the mycelium in the grain spawn and the seeds, allowing more seeds to attach mycelium and efficiently obtain symbiotic nutrients. In addition, the mixing operation should be carried out in a clean and sterile environment (such as a sterile operation table), and sterile glass rods or sterile spoons should be used as tools. The stirring action should be gentle and slow to avoid damaging the seeds or destroying the mycelium structure. Finally, the seeds should be evenly dispersed in the broken grain spawn particles without obvious seed accumulation or grain spawn clumping, ensuring that the seeds can uniformly contact the nutrient source during subsequent culture and improving the consistency of germination. The preparation method of the cultivated species of Coprinus cinereus includes: firstly, preparing sawdust, wheat bran and rice bran raw materials without mildew and impurities, mixing the three raw materials in a clean container, slowly adding water while stirring, and continuously kneading the mixture with hands to adjust the water content until the state of "handing into a ball, no water dripping between fingers, and light kneading scattering" (water content about 60%~65%) is reached, and the preparation of the culture medium is completed; then, the prepared culture medium is loaded into a polypropylene bacteria bag, the loading height is controlled to 2 / 3 of the body of the bacteria bag, the tightness is appropriate, the bag body is not deformed when pinched by hand, then the bag opening is tied with a rope or sealed with a sealing machine, the sealed bacteria bag is placed in a high-pressure sterilization pot, and the bacteria in the culture medium are completely killed under the condition of 121℃ and 0.11MPa for 2.5~3 hours; after sterilization, the bacteria bag is transferred to a clean cooling room and naturally cooled to room temperature (below 25℃) in a sterile environment to avoid contamination by external bacteria during the cooling process; after the bacteria bag is cooled, the inoculation operation is carried out in a sterile inoculation box or a super-clean workbench, 10~15g of the original species is taken from the Coprinus cinereus original species bottle with a sterile inoculation shovel, and is quickly inoculated into the surface of the bacteria bag culture medium, and the bacteria bag is resealed immediately after inoculation; finally, the inoculated bacteria bag is moved to a constant temperature culture room, the temperature is set to 22~26℃, the relative humidity is 60%~70%, and the environment is completely light-proof, the state of the bacteria bag is checked regularly once a week during the period, and the bacteria bag with obvious bacterial contamination (such as green mold and black mold) or abnormal mycelial growth is removed in time, when the mycelium completely covers the surface of the culture medium (about 25~30 days), and there is no obvious bacterial evidence in the bacteria bag, the Coprinus cinereus cultivated species for the symbiotic culture of Cymbidium goeringii seeds is obtained. S3, transparent adhesive bags are selected for easy observation, and the seeds and broken small pieces of Coprinus cinereus mixture are loaded into the bags, where the small pieces of Coprinus cinereus are the broken cultivated species particles; the humidity control is as follows: a calibrated sterile fine mist sprayer is used, the sprayer sprays 0.4mL per press, 2 times on the front and back of the bag opening at a distance of 15~20cm is one round, a total of 3 rounds, the total water volume is 2.4mL, and the bag is shaken gently after each round. After spraying, use a high-precision humidity meter with an accuracy of ±1% to detect the humidity in the bag, the probe should not directly contact the mixture, and the humidity in the bag should reach 60~80%; if it does not meet the standard, spray one more round, if it exceeds the range, unfold the bag opening in a clean environment and stand for 10~20 seconds. After the humidity meets the standard, flatten the bag opening with a sterile sealing clamp, stand it upwards for 2 minutes to balance the moisture, stand to make the water in the bag evenly distributed, mark the groups and the time when the standard is met, and complete the operation. Sealing the bag opening can lock the moisture in the bag, maintain stable humidity, and avoid frequent entry of external air to cause humidity fluctuation, and the bag opening should be sealed without gaps to prevent water loss or bacterial invasion. S4, the purpose of piercing the air hole is to provide oxygen for the seed and fungus on the basis of sealing and moisturizing, to avoid the stagnation of germination caused by lack of oxygen in the bag. 2-5 evenly on both sides can ensure that the air is evenly distributed on both sides of the plastic bag, and avoid uneven air flow caused by single-sided air. The hole diameter of 0.5-1.5cm can meet the oxygen demand of seed and fungus respiration, while also reducing the probability of external bacteria entering through the air hole. When piercing the hole, a sterile punch (or sterilized scissors) should be used to ensure that the hole edge is neat, the plastic bag is not damaged, and the hole position avoids the sealing area of the bag opening and the mixture accumulation area, to ensure the efficiency of air permeation. In this application, preferably 3 holes are evenly pierced on both sides, and the hole diameter is preferably 1cm.

[0019] S5, the light intensity is 0lx, that is, a completely dark environment. Dujuanlan seed germination is sensitive to light, and light will inhibit the activity of seed germination-related enzymes. Complete darkness can make the seed focus on embryo development and symbiosis with fungi. The relative humidity of the external environment is 60-80%, which is consistent with the humidity in the transparent plastic bag, to avoid the risk of water seepage from the inside to the outside due to low external humidity, or the risk of condensation on the surface of the plastic bag due to high external humidity. The temperature is 18-22℃, at which the mycelium of the grain ghost fungus grows actively and can efficiently provide nutrients for the seed. At the same time, the metabolic rate of the seed embryo is moderate, and it will not rot due to high temperature, or germinate slowly due to low temperature. The transparent plastic bag needs to be observed regularly during the cultivation process. The seed starts to germinate after about 18-22 days, and white mycelium is formed after the seed germinates. About 42-48 days form a protocorm with a diameter of 0.5cm, and the germination rate is ≥85%. The relative humidity of the external environment is consistent with the humidity in the transparent plastic bag, and the environmental temperature is preferably controlled at 20℃.

[0020] The present application can start the seed germination and form a protocorm with a diameter of 0.5cm in a short time by mixing the dujuanlan seed with the broken grain ghost fungus cultivation species in a certain proportion and then placing it in a transparent plastic bag, only controlling the completely dark, 18-22℃ constant temperature, 60-80% constant humidity environment and the air permeation on both sides of the plastic bag. The method is simple, low in cost, high in germination efficiency and high in seed germination rate.

[0021] In one possible implementation, the raw materials for preparing the culture medium for the grain ghost fungus cultivation species are sawdust 7 parts, wheat bran 2 parts, and rice chaff 1 part.

[0022] Specifically, when selecting the C. crystallinus cultivation species, the material obtained from the artificial sterile cultivation environment should be selected first to avoid the contamination of the Cymbidium seed by the miscellaneous bacteria in the natural environment and to ensure the cleanliness of the symbiotic process. Sawdust as the main carbon source provides energy support for mycelial growth, wheat bran as the core nitrogen source ensures mycelial protein synthesis and cell proliferation, and rice chaff as an auxiliary nutritional ingredient supplements a small amount of minerals and vitamins. The three are matched in this proportion to avoid the nutritional imbalance caused by the excessive use of a single raw material, ensure the appropriate carbon-nitrogen ratio during mycelial growth, and thus cultivate the C. crystallinus cultivation species with sufficient nutrition and strong mycelial activity, providing a stable nutritional basis for the subsequent symbiosis with the Cymbidium seed.

[0023] In one possible implementation, the air vent is attached with a non-woven fabric patch.

[0024] Specifically, after uniformly tying 2-5 air vents with a hole diameter of 0.5-1.5 cm on both sides of the transparent plastic bag, a non-woven fabric patch should be pasted at each air vent. When pasting, it is necessary to ensure that the non-woven fabric patch completely covers the air vent without wrinkles or gaps. It can not only retain the original air permeability of the air vent, allowing the air inside and outside the transparent plastic bag to circulate moderately, but also effectively block the pollutants such as dust and miscellaneous bacterial spores in the external environment from entering the transparent plastic bag, thereby preventing the mixture from being contaminated and affecting the germination of the Cymbidium seed.

[0025] In one possible implementation, the non-woven fabric patch is a sterile medical-grade non-woven fabric with a grammage of 25-30 g / m 2 , a hole diameter of ≤0.1 mm, and an area that is 2-3 mm larger than the air vent hole diameter to completely cover the air vent and be fixed by a sterile adhesive tape.

[0026] Specifically, the non-woven fabric patch is preferably made of sterile medical-grade polypropylene (polypropylene) or polyester (polyester) non-woven fabric material. This type of material is treated by professional processes such as ethylene oxide sterilization or irradiation sterilization, which can avoid the contamination of the Cymbidium seed and the C. crystallinus cultivation species mixture in the transparent plastic bag from the beginning. At the same time, the grammage of the non-woven fabric patch should be strictly controlled at 25-30 g / m 2The polypropylene or polyester non-woven fabric in this grammage interval has sufficient structural strength, is not easy to break or deform after being pasted, can maintain good air permeability, and will not hinder the air circulation inside and outside the transparent plastic bag due to the thickness of the material. It can fully meet the oxygen supply required for seed and truffle respiration. The aperture of the non-woven fabric paste is ≤0.1mm, relying on the uniform three-dimensional network fiber structure of the polypropylene or polyester non-woven fabric, this aperture can effectively filter the pollutants such as spores and dust particles in the external environment with a diameter of 10-50μm, preventing them from entering the bag through the air-permeable hole and affecting normal air circulation. When cutting the non-woven fabric paste, the area of the non-woven fabric paste should be 2-3mm larger than the aperture of the air-permeable hole (for example, if the aperture of the air-permeable hole is 1cm, the length or diameter of the non-woven fabric paste should be 1.2-1.3cm), to ensure that it completely covers the air-permeable hole and has no edge gaps; sterile adhesive tape is used for fixing, the cut polypropylene or polyester non-woven fabric paste is accurately aligned with the air-permeable hole and covered, then sterile adhesive tape is used to paste along the edge of the non-woven fabric paste, the width of the adhesive tape should be appropriate to wrap the edge of the non-woven fabric paste, and the contact part should be lightly pressed during pasting to ensure that it is tightly pasted without wrinkles or gaps, so as to avoid the non-woven fabric paste falling off or the adhesive tape loosening during cultivation, and to maintain the dual effects of air permeability and pollution prevention, providing a clean and stable environment for the germination of the dwarf iris seed.

[0027] In a possible implementation, the particle size of the truffle cultivation species after crushing is 0.3-0.5cm.

[0028] Specifically, after wearing sterilized latex gloves, the fresh / pre-cultured truffle cultivation species is gently kneaded in a sterile culture dish, and is dispersed into irregular particles of 0.3-0.5cm by the pressure of the finger pads. The force should be controlled during the crushing process, so that the particles can naturally accumulate loosely. The crushed truffle cultivation species can increase the contact area between the truffle and the seed, ensuring efficient supply of symbiotic nutrients, and can maintain the loose state of the mixture, without affecting the subsequent humidity circulation and air permeability.

[0029] In a possible implementation, before mixing the seed with the truffle cultivation species, the seed is wrapped with filter paper and then soaked in a 0.1%-0.3% sodium hypochlorite solution for 5-8min, and then rinsed with sterile distilled water for 3-5 times and the surface water is absorbed.

[0030] Specifically, before the Cymbidium seed is mixed with the C. pruninum cultivation species, the seed needs to be sterilized, and the specific operation is as follows: first, prepare a sodium hypochlorite solution with a mass concentration of 0.1% to 0.3%. The sodium hypochlorite solution with this concentration can effectively kill the bacteria, insect eggs and other pollutants attached to the surface of the seed, avoid cross contamination when mixed with the C. pruninum cultivation species in the future, and will not damage the seed embryo, thus protecting the seed germination potential. The Cymbidium seed taken out is wrapped with filter paper and then put into the solution for 5 to 8 minutes to ensure that each seed can fully contact the solution and improve the sterilization effect. After the soaking is completed, the seed is transferred to a sterile container and repeatedly washed with sterile distilled water for 3 to 5 times to completely remove the residual sodium hypochlorite solution on the surface of the seed, so as to prevent the residual chemical substances from inhibiting the seed germination or affecting the mycelium activity of the C. pruninum. After the washing is completed, the water on the surface of the seed is absorbed with sterile filter paper until the surface of the seed is dry without obvious water stains, so as to avoid the excessive water from causing the seed to be mildewed or diluting the humidity of the subsequent mixture, thus creating clean and suitable conditions for the uniform mixing of the seed and the C. pruninum cultivation species and the subsequent germination.

[0031] In a possible implementation, the transparent adhesive bag is a polyethylene sterile adhesive bag with a thickness of 0.08 to 0.12 mm, the capacity of the transparent adhesive bag is 50 to 100 mL, and the mixture of the seed and the C. pruninum cultivation species accounts for 1 / 3 to 1 / 2 of the volume of the transparent adhesive bag.

[0032] Specifically, the polyethylene material has good transparency, which is convenient for subsequent observation of the state of the mixture in the bag and the generation of the protocorm, and the adhesive bag with the thickness in this range has a certain toughness, which can avoid damage during operation or transportation, and will not affect the transmission of environmental temperature due to the over-thickness of the material, so as to ensure that the temperature in the transparent adhesive bag is stable in the suitable range of 18 to 22℃. At the same time, the capacity of the transparent adhesive bag is 50 to 100 mL, which can meet the demand of the amount of seed for single germination cultivation, and is convenient for flexible placement in the cultivation environment, thus reducing the space occupation. When the mixture is loaded, the amount of the mixture accounts for 1 / 3 to 1 / 2 of the volume of the transparent adhesive bag. If too much is loaded, it will cause the space in the bag to be narrow, the air circulation to be blocked, and the respiration of the seed and the C. pruninum to be affected. If too little is loaded, it will easily cause the humidity to fluctuate too much, which is not conducive to maintaining the stable humidity of 60 to 80%. The amount ratio can ensure the ventilation demand while providing a stable microenvironment for the mixture, thus helping the seed germination. In this application, the transparent adhesive bag is preferably a polyethylene sterile bag with a thickness of 0.1 mm and a capacity of 80 mL, and the loaded mixture accounts for 1 / 3 of the volume of the transparent adhesive bag.

[0033] In a possible implementation, the transparent adhesive bag is turned over once every 7 to 10 days in an environment of 18 to 22℃: the transparent adhesive bag is slowly turned over by 180° from both sides, so that the mixture in the transparent adhesive bag is turned over synchronously with the transparent adhesive bag, and the mixture is kept loose and uniform.

[0034] Specifically, during the culture of Cymbidium seed and the mixture of C. candidum cultivars, a constant environment of 18-22°C needs to be maintained, and the transparent plastic bag is turned over once every 7-10 days. When turning over, both hands hold the two sides of the bag without air holes, keep the hands stable and gentle, slowly rotate the transparent plastic bag by 180°, and ensure that the mixture in the bag can flow naturally after turning over, so as to avoid the mixture clumping or the seeds being squeezed due to turning over too fast or improper force. After turning over, the state of the mixture needs to be observed to keep it loose and evenly distributed, so that each part of the mixture can be in contact with the air in the bag to ensure balanced oxygen supply, and prevent local humidity from being uneven due to long-term contact with the wall of the transparent plastic bag, so as to avoid affecting seed germination due to lack of oxygen or excessive or insufficient humidity, and provide a stable and uniform microenvironment for protocorm generation.

[0035] The present application is further illustrated by the following experiments.

[0036] (1) Cymbidium seed Select 15 mature capsules that meet the requirements of different plant cross-pollination, 170-190 days of growth period, and 2-2.5 cm in length and 0.2-0.3 cm in diameter, with a dark yellow-brown appearance, hard texture, and no tiny cracks. The capsules are divided into 3 groups, 5 capsules in each group.

[0037] Each group of capsules is disinfected with 75% alcohol cotton swabs and then cut open, and all the seeds are poured into a sterile weighing dish. A one-hundredth electronic balance is used to weigh the total weight of the seeds in each group, which is 0.1395g (the same batch of seeds was calibrated in the pre-experiment, with a thousand-grain weight of 0.0046g, and this weight corresponds to the standard seed amount).

[0038] Seed pretreatment: wrap with filter paper, soak in 0.25% sodium hypochlorite solution for 5 minutes, shake gently every 1.5 minutes during the period; then rinse with sterile distilled water for 5 times, dry the water with sterile filter paper, and then place in a constant temperature environment of 20°C for 30 minutes to activate the seed activity, and then store separately for later use.

[0039] (2) C. candidum cultivars Prepare the culture medium according to the ratio of "sawdust 7 parts, wheat bran 2 parts, rice chaff 1 part (mass fraction)", and inoculate the C. candidum spores after sterilization, and culture until the mycelium completely covers the culture medium to obtain the C. candidum cultivars.

[0040] Divide the cultivars into 3 groups, 1 part in each group, with a fresh weight of 450-550g, to ensure that there is no contamination of miscellaneous bacteria, no substrate compaction, and the mycelium is uniform white. Each group of cultivars is crushed to a particle size of 0.3-0.5 cm, immediately placed in a sterile tray, and exposed to air for no more than 5 minutes to prevent the activity of the mycelium from decreasing.

[0041] (3) Containers and tools Prepare 9 0.1mm thick, 80mL polyethylene bags, 3 bags per group, sterilized for 40 minutes; each group is equipped with 1 sterile fine mist spray bottle, calibrated to spray 0.4mL per press; another 10x body microscope, as well as sterile glass rods, tweezers, culture dishes, each group of tools is sterilized and wrapped with sterile tin foil.

[0042] II. Experimental operation steps (I) Mixing and bagging 3 groups of experiments are carried out simultaneously on a sterile operation table. Each group puts the corresponding weight of the Cymbidium seed (0.1395g, corresponding to the total amount of 5 capsule seeds) and the broken crystal grain of Coprinus cultivar (1 part, fresh weight 450~550g) into a sterile glass container, stirs gently in the same direction for 4 minutes, and ensures that the seeds are evenly dispersed in the cultivation medium.

[0043] Fill each polyethylene bag with the mixture, fill the volume of 3 / 8 of the bag. Use a sterile fine mist spray bottle to "spray a little at a time", gently stir the mixture once every 2 sprays, a total of 3 rounds of operation; During the process, use a hygrometer to monitor to ensure that the humidity in the bag reaches 70%±1%. After sealing the bag, place it upside down for 2 minutes to balance the humidity in the bag, and finally mark the group.

[0044] (II) Air hole treatment Make 3 1cm air holes on both sides of each transparent bag, and the hole position is uniformly away from the bag opening and the mixture accumulation area. Paste 30g / m 2 , 0.07mm aperture sterile polypropylene non-woven fabric on each air hole, and then use sterile tape to fully wrap and fix it to ensure no gaps.

[0045] (III) Culture management Put the 9 transparent bags together in the incubator, set the culture conditions to 0lx light, relative humidity 75%±1%, temperature 21℃ constant temperature. Turn over the transparent bags at the same time every day, and gently pat the two sides of the bag after turning over to prevent the mixture from accumulating.

[0046] III. Experimental data (I) Germination start time Record the number of days when 5% of the seeds in each group of 3 transparent bags first appeared white / light color prothallus bud (similar to tiny white spots) on the surface: group 1 for 17 days, group 2 for 16 days, group 3 for 17 days, the average value is 16.7 days, and the germination start time is determined as 17 days.

[0047] (II) Prothallus formation time Statistical days when 30% of the germinated seeds in each group first appeared 0.5cm white prothallus: group 1 for 42 days, group 2 for 43 days, group 3 for 41 days, the average value is 42 days, i.e. the prothallus formation time is 42 days.

[0048] (III) Germination rate After 42 days of culture, 3 transparent adhesive bags in each group were opened, the mixture in the bags was washed clean, and observed under a 10-fold visual microscope. Five-point sampling method (0.5 cm 2 , corresponding to the standard seed amount) was used, and "prosperous protocorm diameter ≥ 0.45 cm" was used as the germination standard to count the germination rate.

[0049] Intra-group average: the germination rates of the three adhesive bags in group 1 were 92.5%, 91.8% and 92.2% respectively, with an average of 92.2%; the germination rates of the three adhesive bags in group 2 were 91.5%, 92.3% and 91.9% respectively, with an average of 91.9%; and the germination rates of the three adhesive bags in group 3 were 92.4%, 92.1% and 92.6% respectively, with an average of 92.4%.

[0050] Total germination rate: the average germination rate of the three groups was 92.2%, with an error of ≤ 0.5%.

[0051] From the above experiment, it can be seen that, by using the present scheme, the Cymbidium seed germination starts at 17 days, the protocorm is formed at 42 days, and the germination rate reaches 92.2%.

[0052] The above is only a preferred embodiment of the present application and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method of promoting seed germination of a Habenaria radiata, the method comprising, The application relates to a method for preparing seeds of a mature Cymbidium ensifolium. Selecting mature Cymbidium ensifolium capsules and taking out seeds in the Cymbidium ensifolium capsules; Mixing the seeds in 5 Cymbidium ensifolium capsules with 1 part of fresh weight 450-550g of broken treated grain agaricus cultivation species; the grain agaricus cultivation species is a solid matrix whole body containing active mycelium formed after sawdust, wheat bran and rice bran are mixed to prepare a culture medium, the culture medium is sterilized, inoculated with grain agaricus spores and cultured until the mycelium completely covers the culture medium; The mixture of the seeds and the grain agaricus cultivation species is put into a transparent adhesive bag, and the humidity is controlled at 60-80%, and the transparent adhesive bag is sealed; 2-5 evenly distributed air holes with a diameter of 0.5-1.5cm are made on both sides of the transparent adhesive bag; The transparent adhesive bag containing the mixture is placed in an environment with an illumination intensity of 0lx, a relative humidity of 60-80% outside the transparent adhesive bag and a temperature of 18-22 DEG C until the original bulbs are generated.

2. The method of facilitating germination of a Goodyera seed according to claim 1, wherein, The raw materials of the culture medium for preparing the grain agaricus cultivation species are mixed in a mass ratio of 7 parts of sawdust, 2 parts of wheat bran and 1 part of rice bran.

3. The method of facilitating germination of a Goodyera seed of claim 1, wherein, The non-woven fabric is attached to the air holes.

4. The method of facilitating germination of a Goodyera seed according to claim 3, wherein, The non-woven fabric patch is a sterile medical grade non-woven fabric, and the grammage is 25-30 g / m 2 The pore size is ≤0.1 mm, and the area of the non-woven fabric patch is 2-3 mm larger than the pore size of the air permeable hole, completely covering the air permeable hole and being fixed by the sterile adhesive tape.

5. The method of facilitating germination of a Goodyera seed of claim 1, wherein, The particle size of the broken grain agaricus cultivation species is 0.3-0.5cm.

6. The method of facilitating germination of a Goodyera seed of claim 1, wherein, Before mixing the seeds with the grain agaricus cultivation species, the seeds are wrapped with filter paper, then soaked in a 0.1%-0.3% sodium hypochlorite solution for 5-8min, washed with sterile distilled water for 3-5 times and the surface water is absorbed.

7. The method of facilitating germination of a Goodyera seed of claim 1, wherein, The transparent adhesive bag is a polyethylene sterile adhesive bag with a thickness of 0.08-0.12mm, the capacity of the transparent adhesive bag is 50-100ml, and the mixture of the seeds and the grain agaricus cultivation species accounts for 1 / 3-1 / 2 of the volume of the transparent adhesive bag.

8. The method of facilitating germination of a Goodyera seed of claim 1, wherein, In the environment with a temperature of 18-22 DEG C, the transparent adhesive bag is turned over once every 7-10 days: the transparent adhesive bag is slowly turned over by 180 DEG on both sides, the mixture in the transparent adhesive bag is turned over synchronously with the transparent adhesive bag, and the mixture is kept loose and uniform.

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