Culture medium breeding method of Bletilla striata and its application

Abstract

The present invention belongs to the field of plant breeding technology, and in particular to a culture medium breeding method and application of Bletilla striata. The present invention discloses a culture medium breeding method of Bletilla striata, and the method comprises the following steps: S1: take mature Bletilla striata seeds, clean and disinfect, then dry and set aside; S2: inoculate Bletilla striata seeds into germination medium, cultivate for 5-8 days to obtain first-generation seedlings; S3: peel off the obtained first-generation seedlings and separate them into single seedlings, inoculate the obtained single seedlings into growth medium and cultivate for 13-18 days to obtain second-generation seedlings; S4: take out the obtained second-generation seedlings from the culture medium, clean and disinfect the roots, then plant them in a culture soil seedling tray, put them into a seedling hardening greenhouse and cultivate for 2-3 months, and finally transplant them to field. The present invention can realize the large-scale breeding of Bletilla striata by preparing a suitable culture medium, so as to expand the planting yield of Bletilla striata.

Description

Technical Field

[0001] The invention belongs to the technical field of plant breeding, and in particular relates to a culture medium breeding method for Bletilla striata and application thereof. Background Art

[0002] Bletilla striata (Thunb. ex A. Murray) Rchb. f., also known as "Baiji", is a herbaceous plant of the genus Bletilla in the Orchidaceae family and a traditional Chinese medicine. Its medicinal part is the dried tuber, which tastes sweet, bitter, astringent, and is cold in nature. It belongs to the stomach meridian, lung meridian, and liver meridian. Bletilla striata has high medicinal value: antibacterial, anti-inflammatory, anti-tumor, immune regulation, and wound healing. Its main components are natural water-soluble polysaccharides, followed by polyphenols and amino acids. At present, the application fields of Bletilla striata involve many aspects, mainly including: traditional Chinese medicine formulas, inclusion materials, bioadhesive materials, hydrogel materials, gene delivery vectors, vascular embolization materials, and polymer micelle materials, etc. It can also be used as anti-freckle cosmetics.

[0003] Bletilla striata is an important Chinese medicinal material with high medicinal value and relatively high market demand. However, due to the low natural reproduction capacity of wild Bletilla striata and long-term harvesting, the number of wild Bletilla striata has decreased sharply and the growth environment has been severely damaged, resulting in a supply shortage of Bletilla striata.

[0004] Therefore, developing a large-scale breeding method for Bletilla striata is still a problem that needs to be solved. Summary of the invention

[0005] The purpose of the present invention is to overcome the problems existing in the prior art and provide a culture medium breeding method and application of Bletilla striata. The present invention can realize large-scale breeding of Bletilla striata by preparing a suitable culture medium, thereby increasing the planting yield of Bletilla striata.

[0006] The purpose of the present invention and the solution to the technical problem are achieved by adopting the following technical solutions.

[0007] One aspect of the present invention provides a culture medium breeding method for Bletilla striata, the method comprising the following steps:

[0008] S1: Take mature Bletilla striata seeds, wash and disinfect them, then dry them for later use;

[0009] S2: inoculating the Bletilla striata seeds into a germination medium and culturing for 5-8 days to obtain first-generation seedlings;

[0010] S3: peeling and separating the first-generation seedlings into single seedlings, inoculating the single seedlings into a growth medium, and culturing for 13-18 days to obtain second-generation seedlings;

[0011] S4: Take out the obtained second-generation seedlings from the culture medium, clean and disinfect the roots, then plant them in the culture soil seedling tray, place them in the seedling greenhouse for cultivation for 2 to 3 months, and finally transplant them;.

[0012] In step S2, the germination medium is composed of: 0.5-1.5 mg / L of plant polysaccharide, 0.1-0.3 mg / L of ginsenoside, 0.5-2 mg / L of glycyrrhizic acid, 4-8 g / L of agar, and the balance is water;

[0013] In step S3, the growth medium composition is: sucrose 5-15 g / L, agar 5-9 g / L, gibberellin 100-300 mg / L, potassium nitrate 200-500 mg / L, ammonium nitrate 500-1000 mg / L, magnesium chloride 100-300 mg / L, calcium chloride 50-100 mg / L, potassium dihydrogen phosphate 100-200 mg / L, urea 20-40 mg / L, sodium chloride 100-300 mg / L, zinc sulfate 5-10 mg / L, copper sulfate 10-20 mg / L, ferrous sulfate 10-30 mg / L, boric acid 2-4 mg / L, potassium iodide 1-3 mg / L, sodium molybdate 0.1-0.3 mg / L, amino acid 0.1-0.3 mg / L, and the balance is water.

[0014] In a preferred embodiment of the present invention, the germination medium is composed of: 0.5-1.5 mg / L of Ulva polysaccharide, 0.1-0.3 mg / L of ginsenoside, 0.5-2 mg / L of glycyrrhizic acid, 4-8 g / L of agar, and the balance is water.

[0015] In a preferred embodiment of the present invention, the germination medium is composed of: 0.5-1.5 mg / L of spirulina polysaccharide, 0.1-0.3 mg / L of ginsenoside, 0.5-2 mg / L of glycyrrhizic acid, 4-8 g / L of agar, and the balance is water.

[0016] In a preferred embodiment of the present invention, the ginsenoside is ginsenoside Rh1.

[0017] In a preferred embodiment of the present invention, in step S2, the culture conditions are: temperature 22-25°C, humidity 60-80%, and light intensity 4000-6000 lux.

[0018] In a preferred embodiment of the present invention, the amino acid is selected from one of glycine, lysine, arginine and histidine, or a mixture of two or more thereof.

[0019] In a preferred embodiment of the present invention, in step S3, the culture conditions are: temperature 22-25°C, humidity 60-80%, and light intensity 4000-6000 lux.

[0020] In a preferred embodiment of the present invention, in step S4, the culture soil is composed of: 100-150 parts of garden soil, 10-30 parts of wood ash, 10-40 parts of rapeseed cake, 5-10 parts of humus soil and 20-40 parts of soybean residue powder, in parts by weight.

[0021] By means of the above technical solution, the present invention has at least the following advantages:

[0022] 1. The present invention cultivates Bletilla striata seeds twice to obtain first-generation seedlings and second-generation seedlings, and finally hardens the second-generation seedlings, so that the survival rate of the obtained Bletilla striata seedlings is significantly increased, the cultivation cycle is shortened, the quality of the Bletilla striata seedlings is improved, and the cost is saved, thereby bringing good economic benefits. The method of the present invention is suitable for the large-scale cultivation of Bletilla striata and has good promotion and utilization value.

[0023] 2. The growth medium of the present invention is mainly composed of plant polysaccharides, ginsenosides and glycyrrhizic acid as the main active substances. Through the synergistic effect of these three substances, the germination rate of Bletilla striata seeds can be significantly improved. Plant polysaccharides such as Ulva polysaccharides and Spirulina polysaccharides can not only provide nutrition for the germination of Bletilla striata seeds, but also have antibacterial and anti-inflammatory effects in synergy with glycyrrhizic acid. Ulva polysaccharides (Ulvan) are the main functional components in Ulva, which are sulfated water-soluble polysaccharides extracted from the cell wall of Ulva, mainly composed of repeating disaccharide units composed of sulfated rhamnose, glucuronic acid, iduronic acid and part of xylose. Ulva polysaccharides have been shown to have a variety of pharmacological properties, such as antioxidant, anticoagulant, anti-tumor, anti-hyperlipidemia and antiviral activities. Spirulina polysaccharide is a water-soluble polysaccharide extracted from spirulina. Many studies have reported that spirulina polysaccharide has a variety of biological activities, such as antiviral, anti-tumor, immunomodulatory, hypoglycemic, and antioxidant effects. Ginsenosides, especially ginsenoside Rh1, have the effect of promoting cell proliferation. These three substances act together on Bletilla striata seeds, which can not only promote the rapid growth and germination of Bletilla striata seeds, but also have antibacterial effects and prevent the influence of the external environment on the growth of Bletilla striata seeds, thereby increasing the germination rate of Bletilla striata seeds and reducing the germination time of Bletilla striata seeds.

[0024] 3. The growth medium of the present invention mainly provides the second-generation seedlings with the nutrients required for growth. In addition to common nutrients such as sucrose and trace elements, gibberellins are also added to promote the growth of the seedlings and improve the growth survival rate of the seedlings. The method of the present invention is simple and easy to operate. The obtained seedlings have good growth and high survival rate after transplanting, and are suitable for large-scale planting.

[0025] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The average plant height changes of each group of seedlings at different times. DETAILED DESCRIPTION

[0027] In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Unless otherwise specified, the percentage content involved in the present invention refers to mass percentage for solid-liquid mixing and solid-solid mixing, and refers to volume percentage for liquid-liquid mixing.

[0029] Unless otherwise specified, the percentage concentrations referred to in the present invention all refer to final concentrations, which refer to the percentage of the added component in the system after the addition of the component.

[0030] The temperature parameters in the present invention, if not specifically limited, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument.

[0031] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0032] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0033] 1. Seed collection and processing

[0034] Harvest mature Bletilla striata seeds, gently rub off the peel with your hands, remove impurities and shriveled seeds from the Bletilla striata seeds and set aside, select seeds with uniform size, full grains and uniform color, wash the Bletilla striata seeds with deionized water, select the full seeds that sink after rinsing the seeds with water, then disinfect them with a 5% volume fraction of carbendazim aqueous solution for 10 min, then rinse with deionized water until odorless, and then dry with absorbent paper for set aside.

[0035] 2. Preparation of Ulva polysaccharide

[0036] Weigh the ulva powder, and mix the ulva powder with water at a mass volume ratio of 1g:40mL. Then, ultrasonic assisted extraction was performed at 60℃, 50kHz, and 200W power for 120min, and then the supernatant was taken after centrifugation at 5000rpm for 10min. A 95% ethanol solution was added to the obtained supernatant at a volume ratio of 1:4, and the precipitate was placed at 50℃ and dried to obtain extract I. The extract I was dissolved in water at a mass volume ratio of 1g:100mL, and protease was added to the solution to make the enzyme activity 250U / mL. After enzymatic hydrolysis at 40℃ for 150min, the enzyme activity was inactivated at 100℃ for 20min, and the supernatant was taken after centrifugation. It was dialyzed for 48h using a 10kDa dialysis bag and concentrated and freeze-dried to obtain ulva polysaccharide.

[0037] 3. Preparation of Spirulina Polysaccharide

[0038] Weigh the spirulina powder, and mix the spirulina powder and water evenly at a mass volume ratio of 1g:40mL. Then, ultrasonic extraction was performed at 60℃, 50kHz, and 200W for 120min, and then the supernatant was taken after centrifugation at 5000rpm for 10min. A 95% ethanol solution was added to the obtained supernatant at a volume ratio of 1:4, and the supernatant was precipitated at 4℃ overnight, and then the precipitate was centrifuged and placed at 50℃ to obtain a crude extract I. The extract I was dissolved in water at a mass volume ratio of 1g:100mL, and protease was added to the solution to make the enzyme activity 250U / mL, and after enzymatic hydrolysis at 40℃ for 150min, the enzyme activity was inactivated at 100℃ for 20min, and the supernatant was taken after centrifugation, and then dialyzed for 48h using a 10kDa dialysis bag, and then concentrated and freeze-dried to obtain spirulina polysaccharide.

[0039] 4. Preparation of culture soil

[0040] According to weight, 120 parts of farmland soil, 20 parts of wood ash, 30 parts of rapeseed cake, 10 parts of humus soil and 30 parts of soybean residue powder are mixed evenly.

[0041] Example 1

[0042] (1) Preparation of germination medium: Add each component to water in sequence to make the concentration of each component as follows: 1.0 mg / L of Ulva polysaccharide, 0.2 mg / L of ginsenoside Rh1, 1.25 mg / L of glycyrrhizic acid, and 6 g / L of agar, followed by sterilization at 120°C for use.

[0043] (2) Preparation of growth medium: Add each component to water in sequence to make the concentration of each component as follows: sucrose 10 g / L, agar 7 g / L, gibberellin 200 mg / L, potassium nitrate 350 mg / L, ammonium nitrate 750 mg / L, magnesium chloride 200 mg / L, calcium chloride 75 mg / L, potassium dihydrogen phosphate 150 mg / L, urea 30 mg / L, sodium chloride 200 mg / L, zinc sulfate 7.5 mg / L, copper sulfate 15 mg / L, ferrous sulfate 20 mg / L, boric acid 3 mg / L, potassium iodide 2 mg / L, sodium molybdate 0.2 mg / L, amino acid (a mixture of glycine, lysine, arginine in equal mass ratios) 0.2 mg / L, then sterilize at 120°C for use.

[0044] (3) Culture medium breeding of Bletilla striata: 200 seeds of Bletilla striata were inoculated into germination medium and cultured for 7 days at a temperature of 25°C, a humidity of 70%, and a light intensity of 5000 lux to obtain the first generation of seedlings. The germination of the seeds was statistically analyzed, and the results are shown in Table 1.

[0045] The first generation seedlings were separated into single seedlings, and the single seedlings were inoculated into the growth medium and cultured for 15 days at a temperature of 25°C, a humidity of 70%, and a light intensity of 5000 lux to obtain the second generation seedlings. The second generation seedlings were taken out of the culture medium, the roots were cleaned and disinfected, and then 50 second generation seedlings with basically the same growth were selected and planted in the culture soil seedling tray, placed in the seedling hardening greenhouse for cultivation for 2 months, and finally transplanted to the field. The average plant height of the seedlings was counted every 10 days during the hardening period. The results are shown in Figure 1 .

[0046] Example 2

[0047] (1) Preparation of germination medium: Add each component to water in sequence to make the concentration of each component as follows: 0.5 mg / L ulva polysaccharide, 0.3 mg / L ginsenoside Rh1, 2 mg / L glycyrrhizic acid, 4 g / L agar, then sterilize at 120°C for use.

[0048] (2) Preparation of growth medium: Add each component to water in sequence to make the concentration of each component as follows: sucrose 5 g / L, agar 9 g / L, gibberellin 100 mg / L, potassium nitrate 500 mg / L, ammonium nitrate 500 mg / L, magnesium chloride 300 mg / L, calcium chloride 50 mg / L, potassium dihydrogen phosphate 200 mg / L, urea 20 mg / L, sodium chloride 300 mg / L, zinc sulfate 5 mg / L, copper sulfate 20 mg / L, ferrous sulfate 10 mg / L, boric acid 4 mg / L, potassium iodide 1 mg / L, sodium molybdate 0.3 mg / L, amino acid (a mixture of glycine, lysine, histidine in equal proportions) 0.1 mg / L, then sterilize at 120°C for use.

[0049] (3) Culture medium breeding of Bletilla striata: 200 seeds of Bletilla striata were inoculated into germination medium and cultured for 7 days at a temperature of 25°C, a humidity of 60%, and a light intensity of 4000 lux to obtain the first generation of seedlings. The germination of the seeds was statistically analyzed, and the results are shown in Table 1.

[0050] The first generation seedlings were separated into single seedlings, and the single seedlings were inoculated into a growth medium and cultured for 15 days at a temperature of 25°C, a humidity of 60%, and a light intensity of 4000 lux to obtain second generation seedlings. The second generation seedlings were taken out from the culture medium, the roots were cleaned and disinfected, and then 50 second generation seedlings with basically the same growth were selected and planted in a culture soil seedling tray, placed in a seedling hardening greenhouse for cultivation for 2 months (the cultivation conditions were consistent with those in Example 1), and finally transplanted to the field. The average plant height of the seedlings was counted every 10 days during the hardening period, and the results were shown in Table 1. Figure 1 .

[0051] Example 3

[0052] (1) Preparation of germination medium: Add each component to water in sequence to make the concentration of each component as follows: 1.5 mg / L of Ulva polysaccharide, 0.1 mg / L of ginsenoside Rh1, 0.5 mg / L of glycyrrhizic acid, and 8 g / L of agar, followed by sterilization at 120°C for use.

[0053] (2) Preparation of growth medium: Add each component to water in sequence to make the concentration of each component as follows: sucrose 15 g / L, agar 5 g / L, gibberellin 300 mg / L, potassium nitrate 200 mg / L, ammonium nitrate 1000 mg / L, magnesium chloride 100 mg / L, calcium chloride 100 mg / L, potassium dihydrogen phosphate 100 mg / L, urea 40 mg / L, sodium chloride 100 mg / L, zinc sulfate 10 mg / L, copper sulfate 10 mg / L, ferrous sulfate 30 mg / L, boric acid 2 mg / L, potassium iodide 3 mg / L, sodium molybdate 0.1 mg / L, amino acid (a mixture of glycine, lysine, arginine, histidine in equal mass ratios) 0.3 mg / L, then sterilize at 120°C for use.

[0054] (3) Culture medium breeding of Bletilla striata: 200 seeds of Bletilla striata were inoculated into germination medium and cultured for 7 days at a temperature of 25°C, a humidity of 80%, and a light intensity of 6000 lux to obtain the first generation of seedlings. The germination of the seeds was statistically analyzed, and the results are shown in Table 1.

[0055] The first generation seedlings were separated into single seedlings, and the single seedlings were inoculated into a growth medium and cultured for 15 days at a temperature of 25°C, a humidity of 80%, and a light intensity of 6000 lux to obtain second generation seedlings. The second generation seedlings were taken out from the culture medium, the roots were cleaned and disinfected, and then 50 second generation seedlings with basically the same growth were selected and planted in a culture soil seedling tray, placed in a seedling hardening greenhouse for cultivation for 2 months (the cultivation conditions were consistent with those in Example 1), and finally transplanted to the field. The average plant height of the seedlings was counted every 10 days during the hardening period, and the results were shown in Table 1. Figure 1 .

[0056] Example 4

[0057] (1) Preparation of germination medium: Add each component to water in sequence to make the concentration of each component as follows: spirulina polysaccharide 1.0 mg / L, ginsenoside Rh1 0.2 mg / L, glycyrrhizic acid 1.25 mg / L, agar 6 g / L, then sterilize at 120°C for use.

[0058] (2) Preparation of growth medium: Add each component to water in sequence to make the concentration of each component as follows: sucrose 10 g / L, agar 7 g / L, gibberellin 200 mg / L, potassium nitrate 350 mg / L, ammonium nitrate 750 mg / L, magnesium chloride 200 mg / L, calcium chloride 75 mg / L, potassium dihydrogen phosphate 150 mg / L, urea 30 mg / L, sodium chloride 200 mg / L, zinc sulfate 7.5 mg / L, copper sulfate 15 mg / L, ferrous sulfate 20 mg / L, boric acid 3 mg / L, potassium iodide 2 mg / L, sodium molybdate 0.2 mg / L, amino acid (a mixture of glycine, lysine, arginine in equal mass ratios) 0.2 mg / L, then sterilize at 120°C for use.

[0059] (3) Culture medium breeding of Bletilla striata: 200 seeds of Bletilla striata were inoculated into germination medium and cultured for 7 days at a temperature of 25°C, a humidity of 70%, and a light intensity of 5000 lux to obtain the first generation of seedlings. The germination of the seeds was statistically analyzed, and the results are shown in Table 1.

[0060] The first generation seedlings were separated into single seedlings, and the single seedlings were inoculated into a growth medium and cultured for 15 days at a temperature of 25°C, a humidity of 70%, and a light intensity of 5000 lux to obtain second generation seedlings. The second generation seedlings were taken out from the culture medium, the roots were cleaned and disinfected, and then 50 second generation seedlings with basically the same growth were selected and planted in a culture soil seedling tray, placed in a seedling hardening greenhouse for cultivation for 2 months (the cultivation conditions were consistent with those in Example 1), and finally transplanted to the field. The average plant height of the seedlings was counted every 10 days during the hardening period, and the results were shown in Table 1. Figure 1 .

[0061] Example 5

[0062] (1) Preparation of germination medium: Add each component to water in sequence to make the concentration of each component as follows: spirulina polysaccharide 0.5 mg / L, ginsenoside Rh1 0.3 mg / L, glycyrrhizic acid 2 mg / L, agar 4 g / L, then sterilize at 120°C for use.

[0063] (2) Preparation of growth medium: Add each component to water in sequence to make the concentration of each component as follows: sucrose 5 g / L, agar 9 g / L, gibberellin 100 mg / L, potassium nitrate 500 mg / L, ammonium nitrate 500 mg / L, magnesium chloride 300 mg / L, calcium chloride 50 mg / L, potassium dihydrogen phosphate 200 mg / L, urea 20 mg / L, sodium chloride 300 mg / L, zinc sulfate 5 mg / L, copper sulfate 20 mg / L, ferrous sulfate 10 mg / L, boric acid 4 mg / L, potassium iodide 1 mg / L, sodium molybdate 0.3 mg / L, amino acid (a mixture of glycine, lysine, histidine in equal proportions) 0.1 mg / L, then sterilize at 120°C for use.

[0064] (3) Culture medium breeding of Bletilla striata: 200 seeds of Bletilla striata were inoculated into germination medium and cultured for 7 days at a temperature of 25°C, a humidity of 60%, and a light intensity of 4000 lux to obtain the first generation of seedlings. The germination of the seeds was statistically analyzed, and the results are shown in Table 1.

[0065] The first generation seedlings were separated into single seedlings, and the single seedlings were inoculated into a growth medium and cultured for 15 days at a temperature of 25°C, a humidity of 60%, and a light intensity of 4000 lux to obtain second generation seedlings. The second generation seedlings were taken out from the culture medium, the roots were cleaned and disinfected, and then 50 second generation seedlings with basically the same growth were selected and planted in a culture soil seedling tray, placed in a seedling hardening greenhouse for cultivation for 2 months (the cultivation conditions were consistent with those in Example 1), and finally transplanted to the field. The average plant height of the seedlings was counted every 10 days during the hardening period, and the results were shown in Table 1. Figure 1 .

[0066] Example 6

[0067] (1) Preparation of germination medium: Add each component to water in sequence to make the concentration of each component as follows: spirulina polysaccharide 1.5 mg / L, ginsenoside Rh1 0.1 mg / L, glycyrrhizic acid 0.5 mg / L, agar 8 g / L, then sterilize at 120°C for use.

[0068] (2) Preparation of growth medium: Add each component to water in sequence to make the concentration of each component as follows: sucrose 15 g / L, agar 5 g / L, gibberellin 300 mg / L, potassium nitrate 200 mg / L, ammonium nitrate 1000 mg / L, magnesium chloride 100 mg / L, calcium chloride 100 mg / L, potassium dihydrogen phosphate 100 mg / L, urea 40 mg / L, sodium chloride 100 mg / L, zinc sulfate 10 mg / L, copper sulfate 10 mg / L, ferrous sulfate 30 mg / L, boric acid 2 mg / L, potassium iodide 3 mg / L, sodium molybdate 0.1 mg / L, amino acid (a mixture of glycine, lysine, arginine, histidine in equal mass ratios) 0.3 mg / L, then sterilize at 120°C for use.

[0069] (3) Culture medium breeding of Bletilla striata: 200 seeds of Bletilla striata were inoculated into germination medium and cultured for 7 days at a temperature of 25°C, a humidity of 80%, and a light intensity of 6000 lux to obtain the first generation of seedlings. The germination of the seeds was statistically analyzed, and the results are shown in Table 1.

[0070] The first generation seedlings were separated into single seedlings, and the single seedlings were inoculated into a growth medium and cultured for 15 days at a temperature of 25°C, a humidity of 80%, and a light intensity of 6000 lux to obtain second generation seedlings. The second generation seedlings were taken out from the culture medium, the roots were cleaned and disinfected, and then 50 second generation seedlings with basically the same growth were selected and planted in a culture soil seedling tray, placed in a seedling hardening greenhouse for cultivation for 2 months (the cultivation conditions were consistent with those in Example 1), and finally transplanted to the field. The average plant height of the seedlings was counted every 10 days during the hardening period, and the results were shown in Table 1. Figure 1 .

[0071] Comparative Example 1

[0072] (1) Preparation of germination medium: Add each component to water in sequence to make the concentration of each component: ginsenoside Rh 10.2 mg / L, glycyrrhizic acid 1.25 mg / L, agar 6 g / L, then sterilize at 120°C for use.

[0073] (2) Preparation of growth medium: Add each component to water in sequence to make the concentration of each component as follows: sucrose 10 g / L, agar 7 g / L, gibberellin 200 mg / L, potassium nitrate 350 mg / L, ammonium nitrate 750 mg / L, magnesium chloride 200 mg / L, calcium chloride 75 mg / L, potassium dihydrogen phosphate 150 mg / L, urea 30 mg / L, sodium chloride 200 mg / L, zinc sulfate 7.5 mg / L, copper sulfate 15 mg / L, ferrous sulfate 20 mg / L, boric acid 3 mg / L, potassium iodide 2 mg / L, sodium molybdate 0.2 mg / L, amino acid (a mixture of glycine, lysine, arginine in equal mass ratios) 0.2 mg / L, then sterilize at 120°C for use.

[0074] (3) Culture medium breeding of Bletilla striata: 200 seeds of Bletilla striata were inoculated into germination medium and cultured for 7 days at a temperature of 25°C, a humidity of 70%, and a light intensity of 5000 lux to obtain the first generation of seedlings. The germination of the seeds was statistically analyzed, and the results are shown in Table 1.

[0075] The first generation seedlings were separated into single seedlings, and the single seedlings were inoculated into a growth medium and cultured for 15 days at a temperature of 25°C, a humidity of 70%, and a light intensity of 5000 lux to obtain second generation seedlings. The second generation seedlings were taken out from the culture medium, the roots were cleaned and disinfected, and then 50 second generation seedlings with basically the same growth were selected and planted in a culture soil seedling tray, placed in a seedling hardening greenhouse for cultivation for 2 months (the cultivation conditions were consistent with those in Example 1), and finally transplanted to the field. The average plant height of the seedlings was counted every 10 days during the hardening period, and the results were shown in Table 1. Figure 1 .

[0076] Comparative Example 2

[0077] (1) Preparation of germination medium: Add each component to water in sequence to make the concentration of each component: 1.0 mg / L of Ulva polysaccharide, 1.25 mg / L of glycyrrhizic acid, and 6 g / L of agar, followed by sterilization at 120°C for use.

[0078] (2) Preparation of growth medium: Add each component to water in sequence to make the concentration of each component as follows: sucrose 10 g / L, agar 7 g / L, gibberellin 200 mg / L, potassium nitrate 350 mg / L, ammonium nitrate 750 mg / L, magnesium chloride 200 mg / L, calcium chloride 75 mg / L, potassium dihydrogen phosphate 150 mg / L, urea 30 mg / L, sodium chloride 200 mg / L, zinc sulfate 7.5 mg / L, copper sulfate 15 mg / L, ferrous sulfate 20 mg / L, boric acid 3 mg / L, potassium iodide 2 mg / L, sodium molybdate 0.2 mg / L, amino acid (a mixture of glycine, lysine, arginine in equal mass ratios) 0.2 mg / L, then sterilize at 120°C for use.

[0079] (3) Culture medium breeding of Bletilla striata: 200 seeds of Bletilla striata were inoculated into germination medium and cultured for 7 days at a temperature of 25°C, a humidity of 70%, and a light intensity of 5000 lux to obtain the first generation of seedlings. The germination of the seeds was statistically analyzed, and the results are shown in Table 1.

[0080] The first generation seedlings were separated into single seedlings, and the single seedlings were inoculated into a growth medium and cultured for 15 days at a temperature of 25°C, a humidity of 70%, and a light intensity of 5000 lux to obtain second generation seedlings. The second generation seedlings were taken out from the culture medium, the roots were cleaned and disinfected, and then 50 second generation seedlings with basically the same growth were selected and planted in a culture soil seedling tray, placed in a seedling hardening greenhouse for cultivation for 2 months (the cultivation conditions were consistent with those in Example 1), and finally transplanted to the field. The average plant height of the seedlings was counted every 10 days during the hardening period, and the results were shown in Table 1. Figure 1 .

[0081] Comparative Example 3

[0082] (1) Preparation of germination medium: Add each component to water in sequence to make the concentration of each component as follows: 1.0 mg / L of Ulva polysaccharide, 0.2 mg / L of ginsenoside Rh1, and 6 g / L of agar, followed by sterilization at 120°C for use.

[0083] (2) Preparation of growth medium: Add each component to water in sequence to make the concentration of each component as follows: sucrose 10 g / L, agar 7 g / L, gibberellin 200 mg / L, potassium nitrate 350 mg / L, ammonium nitrate 750 mg / L, magnesium chloride 200 mg / L, calcium chloride 75 mg / L, potassium dihydrogen phosphate 150 mg / L, urea 30 mg / L, sodium chloride 200 mg / L, zinc sulfate 7.5 mg / L, copper sulfate 15 mg / L, ferrous sulfate 20 mg / L, boric acid 3 mg / L, potassium iodide 2 mg / L, sodium molybdate 0.2 mg / L, amino acid (a mixture of glycine, lysine, arginine in equal mass ratios) 0.2 mg / L, then sterilize at 120°C for use.

[0084] (3) Culture medium breeding of Bletilla striata: 200 seeds of Bletilla striata were inoculated into germination medium and cultured for 7 days at a temperature of 25°C, a humidity of 70%, and a light intensity of 5000 lux to obtain the first generation of seedlings. The germination of the seeds was statistically analyzed, and the results are shown in Table 1.

[0085] The first generation seedlings were separated into single seedlings, and the single seedlings were inoculated into a growth medium and cultured for 15 days at a temperature of 25°C, a humidity of 70%, and a light intensity of 5000 lux to obtain second generation seedlings. The second generation seedlings were taken out from the culture medium, the roots were cleaned and disinfected, and then 50 second generation seedlings with basically the same growth were selected and planted in a culture soil seedling tray, placed in a seedling hardening greenhouse for cultivation for 2 months (the cultivation conditions were consistent with those in Example 1), and finally transplanted to the field. The average plant height of the seedlings was counted every 10 days during the hardening period, and the results were shown in Table 1. Figure 1 .

[0086] Comparative Example 4

[0087] (1) Preparation of germination medium: Add each component to water in sequence to make the concentration of each component: glycyrrhizic acid 1.25 mg / L, agar 6 g / L, then sterilize at 120°C for use.

[0088] (2) Preparation of growth medium: Add each component to water in sequence to make the concentration of each component as follows: sucrose 10 g / L, agar 7 g / L, gibberellin 200 mg / L, potassium nitrate 350 mg / L, ammonium nitrate 750 mg / L, magnesium chloride 200 mg / L, calcium chloride 75 mg / L, potassium dihydrogen phosphate 150 mg / L, urea 30 mg / L, sodium chloride 200 mg / L, zinc sulfate 7.5 mg / L, copper sulfate 15 mg / L, ferrous sulfate 20 mg / L, boric acid 3 mg / L, potassium iodide 2 mg / L, sodium molybdate 0.2 mg / L, amino acid (a mixture of glycine, lysine, arginine in equal mass ratios) 0.2 mg / L, then sterilize at 120°C for use.

[0089] (3) Culture medium breeding of Bletilla striata: 200 seeds of Bletilla striata were inoculated into germination medium and cultured for 7 days at a temperature of 25°C, a humidity of 70%, and a light intensity of 5000 lux to obtain the first generation of seedlings. The germination of the seeds was statistically analyzed, and the results are shown in Table 1.

[0090] The first generation seedlings were separated into single seedlings, and the single seedlings were inoculated into a growth medium and cultured for 15 days at a temperature of 25°C, a humidity of 70%, and a light intensity of 5000 lux to obtain second generation seedlings. The second generation seedlings were taken out from the culture medium, the roots were cleaned and disinfected, and then 50 second generation seedlings with basically the same growth were selected and planted in a culture soil seedling tray, placed in a seedling hardening greenhouse for cultivation for 2 months (the cultivation conditions were consistent with those in Example 1), and finally transplanted to the field. The average plant height of the seedlings was counted every 10 days during the hardening period, and the results were shown in Table 1. Figure 1 .

[0091] Comparative Example 5

[0092] (1) Preparation of germination medium: Add each component to water in sequence to make the concentration of each component: 1.0 mg / L of Ulva polysaccharide and 6 g / L of agar, then sterilize at 120°C for use.

[0093] (2) Preparation of growth medium: Add each component to water in sequence to make the concentration of each component as follows: sucrose 10 g / L, agar 7 g / L, gibberellin 200 mg / L, potassium nitrate 350 mg / L, ammonium nitrate 750 mg / L, magnesium chloride 200 mg / L, calcium chloride 75 mg / L, potassium dihydrogen phosphate 150 mg / L, urea 30 mg / L, sodium chloride 200 mg / L, zinc sulfate 7.5 mg / L, copper sulfate 15 mg / L, ferrous sulfate 20 mg / L, boric acid 3 mg / L, potassium iodide 2 mg / L, sodium molybdate 0.2 mg / L, amino acid (a mixture of glycine, lysine, arginine in equal mass ratios) 0.2 mg / L, then sterilize at 120°C for use.

[0094] (3) Culture medium breeding of Bletilla striata: 200 seeds of Bletilla striata were inoculated into germination medium and cultured for 7 days at a temperature of 25°C, a humidity of 70%, and a light intensity of 5000 lux to obtain the first generation of seedlings. The germination of the seeds was statistically analyzed, and the results are shown in Table 1.

[0095] The first generation seedlings were separated into single seedlings, and the single seedlings were inoculated into a growth medium and cultured for 15 days at a temperature of 25°C, a humidity of 70%, and a light intensity of 5000 lux to obtain second generation seedlings. The second generation seedlings were taken out from the culture medium, the roots were cleaned and disinfected, and then 50 second generation seedlings with basically the same growth were selected and planted in a culture soil seedling tray, placed in a seedling hardening greenhouse for cultivation for 2 months (the cultivation conditions were consistent with those in Example 1), and finally transplanted to the field. The average plant height of the seedlings was counted every 10 days during the hardening period, and the results were shown in Table 1. Figure 1 .

[0096] Comparative Example 6

[0097] (1) Preparation of germination medium: Add each component to water in sequence to make the concentration of each component: ginsenoside Rh 10.2 mg / L, agar 6 g / L, then sterilize at 120°C for use.

[0098] (2) Preparation of growth medium: Add each component to water in sequence to make the concentration of each component as follows: sucrose 10 g / L, agar 7 g / L, gibberellin 200 mg / L, potassium nitrate 350 mg / L, ammonium nitrate 750 mg / L, magnesium chloride 200 mg / L, calcium chloride 75 mg / L, potassium dihydrogen phosphate 150 mg / L, urea 30 mg / L, sodium chloride 200 mg / L, zinc sulfate 7.5 mg / L, copper sulfate 15 mg / L, ferrous sulfate 20 mg / L, boric acid 3 mg / L, potassium iodide 2 mg / L, sodium molybdate 0.2 mg / L, amino acid (a mixture of glycine, lysine, arginine in equal mass ratios) 0.2 mg / L, then sterilize at 120°C for use.

[0099] (3) Culture medium breeding of Bletilla striata: 200 seeds of Bletilla striata were inoculated into germination medium and cultured for 7 days at a temperature of 25°C, a humidity of 70%, and a light intensity of 5000 lux to obtain the first generation of seedlings. The germination of the seeds was statistically analyzed, and the results are shown in Table 1.

[0100] The first generation seedlings were separated into single seedlings, and the single seedlings were inoculated into a growth medium and cultured for 15 days at a temperature of 25°C, a humidity of 70%, and a light intensity of 5000 lux to obtain second generation seedlings. The second generation seedlings were taken out from the culture medium, the roots were cleaned and disinfected, and then 50 second generation seedlings with basically the same growth were selected and planted in a culture soil seedling tray, placed in a seedling hardening greenhouse for cultivation for 2 months (the cultivation conditions were consistent with those in Example 1), and finally transplanted to the field. The average plant height of the seedlings was counted every 10 days during the hardening period, and the results were shown in Table 1. Figure 1 .

[0101] Comparative Example 7

[0102] (1) Preparation of germination medium: Add each component to water in sequence to make the concentration of each component: agar 6 g / L, then sterilize at 120°C for use.

[0103] (2) Preparation of growth medium: Add each component to water in sequence to make the concentration of each component as follows: sucrose 10 g / L, agar 7 g / L, gibberellin 200 mg / L, potassium nitrate 350 mg / L, ammonium nitrate 750 mg / L, magnesium chloride 200 mg / L, calcium chloride 75 mg / L, potassium dihydrogen phosphate 150 mg / L, urea 30 mg / L, sodium chloride 200 mg / L, zinc sulfate 7.5 mg / L, copper sulfate 15 mg / L, ferrous sulfate 20 mg / L, boric acid 3 mg / L, potassium iodide 2 mg / L, sodium molybdate 0.2 mg / L, amino acid (a mixture of glycine, lysine, arginine in equal mass ratios) 0.2 mg / L, then sterilize at 120°C for use.

[0104] (3) Culture medium breeding of Bletilla striata: 200 seeds of Bletilla striata were inoculated into germination medium and cultured for 7 days at a temperature of 25°C, a humidity of 70%, and a light intensity of 5000 lux to obtain the first generation of seedlings. The germination of the seeds was statistically analyzed, and the results are shown in Table 1.

[0105] The first generation seedlings were separated into single seedlings, and the single seedlings were inoculated into a growth medium and cultured for 15 days at a temperature of 25°C, a humidity of 70%, and a light intensity of 5000 lux to obtain second generation seedlings. The second generation seedlings were taken out from the culture medium, the roots were cleaned and disinfected, and then 50 second generation seedlings with basically the same growth were selected and planted in a culture soil seedling tray, placed in a seedling hardening greenhouse for cultivation for 2 months (the cultivation conditions were consistent with those in Example 1), and finally transplanted to the field. The average plant height of the seedlings was counted every 10 days during the hardening period, and the results were shown in Table 1. Figure 1 .

[0106] Germination rate = number of germinations at time t / total number of seeds × 100%;

[0107] Average seedling height = total height of all seedlings / number of seedlings.

[0108] Table 1 Statistics of germination of Bletilla striata seeds in different groups

[0109] Group Number of germination / grain Germination rate / % Average seedling height / cm Example 1 189 94.5 1.66 Example 2 185 92.5 1.65 Example 3 191 95.5 1.67 Example 4 178 89 1.65 Example 5 172 86 1.64 Example 6 181 90.5 1.67 Comparative Example 1 165 82.5 1.63 Comparative Example 2 151 75.5 1.56 Comparative Example 3 170 85 1.62 Comparative Example 4 132 66 1.51 Comparative Example 5 144 72 1.54 Comparative Example 6 149 74.5 1.57 Comparative Example 7 119 59.5 1.48

[0110] From the results in Table 1, it can be seen that the germination rates of the Bletilla striata seeds of Examples 1-6 of the present invention are significantly higher than those of Comparative Examples 1-7, which indicates that Ulva polysaccharide / Spirulina polysaccharide can synergistically act with ginsenoside Rh1 glycyrrhizic acid to promote the germination of Bletilla striata seeds. In addition, the first-generation seedlings obtained from the germination of the Bletilla striata seeds of Examples 1-6 grow well.

[0111] Depend on Figure 1 It can be seen from the results that within 2 months of hardening and cultivation, the plant height of the seedlings in each group of embodiments showed an increasing trend, and the growth rate of the seedlings in embodiments 1-6 was significantly higher than that in comparative examples 1-7, which indicates that under the same cultivation conditions, the method of the present invention can obtain seedlings with better growth.

[0112] Experiment 1: Survival of transplanted seedlings

[0113] 100 Bletilla striata seedlings with substantially the same growth potential after being cultivated by the methods of Examples 1 and 4 and Comparative Examples 1 to 7 were selected as seedlings to be transplanted.

[0114] A large field of fruit and vegetable planting base was selected and divided into 9 experimental fields according to their area size, which were numbered 1 to 9.

[0115] The seedlings of Examples 1, 4 and Comparative Examples 1 to 7 were transplanted into Experimental Fields 1 to 9, respectively. After 15 days, the number of surviving seedlings in each experimental field was counted and the survival rate was calculated. The results are shown in Table 2. During the planting period, each experimental field maintained the same field management mode.

[0116] Table 2 Seedling survival rate in different experimental fields

[0117] Group Survival rate / % Example 1 100 Example 4 99 Comparative Example 1 97 Comparative Example 2 95 Comparative Example 3 98 Comparative Example 4 93 Comparative Example 5 94 Comparative Example 6 96 Comparative Example 7 91

[0118] It can be seen from the results in Table 2 that the seedling transplant survival rate obtained in each group of embodiments can be maintained at more than 90%, and the seedling transplant survival rate after hardening and cultivation is not much different, but overall, the seedling transplant survival rate of embodiments 1 and 4 is higher.

[0119] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of equivalent changes by using the methods and technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A culture medium breeding method for Bletilla striata, characterized in that: The method comprises the following steps: S1: Take mature Bletilla striata seeds, wash and disinfect them, then dry them for later use; S2: inoculating the Bletilla striata seeds into a germination medium and culturing for 5-8 days to obtain first-generation seedlings; S3: peeling and separating the first-generation seedlings into single seedlings, inoculating the single seedlings into a growth medium, and culturing for 13-18 days to obtain second-generation seedlings; S4: The obtained second-generation seedlings are taken out from the culture medium, the roots are cleaned and disinfected, and then planted in a culture soil seedling tray, placed in a seedling hardening greenhouse for 2-3 months, and finally transplanted; In step S2, the germination medium is composed of: 0.5-1.5 mg / L of plant polysaccharide, 0.1-0.3 mg / L of ginsenoside, 0.5-2 mg / L of glycyrrhizic acid, 4-8 g / L of agar, and the balance is water; In step S3, the growth medium composition is: sucrose 5-15 g / L, agar 5-9 g / L, gibberellin 100-300 mg / L, potassium nitrate 200-500 mg / L, ammonium nitrate 500-1000 mg / L, magnesium chloride 100-300 mg / L, calcium chloride 50-100 mg / L, potassium dihydrogen phosphate 100-200 mg / L, urea 20-40 mg / L, sodium chloride 100-300 mg / L, zinc sulfate 5-10 mg / L, copper sulfate 10-20 mg / L, ferrous sulfate 10-30 mg / L, boric acid 2-4 mg / L, potassium iodide 1-3 mg / L, sodium molybdate 0.1-0.3 mg / L, amino acid 0.1-0.3 mg / L, and the balance is water; The ginsenoside is ginsenoside Rh1; The plant polysaccharide is ulva polysaccharide or spirulina polysaccharide; The preparation of the ulva polysaccharide comprises the following steps: weighing ulva powder, and uniformly mixing the ulva powder with water at a mass volume ratio of 1g:40mL; then performing ultrasonic assisted extraction at 60°C, 50kHz, and 200W power for 120min, and then centrifuging at 5000rpm for 10min to obtain a supernatant, adding a 95% ethanol solution by volume to the obtained supernatant at a volume ratio of 1:4, and precipitating the solution at 4°C overnight, and then centrifuging to obtain a precipitate, and drying the precipitate at 50°C to obtain an extract I; dissolving the extract I in water at a mass volume ratio of 1g:100mL to obtain a solution, adding a protease to the solution so that the enzyme activity is 250U / mL, and performing enzymolysis at 40°C for 150min, and then inactivating the enzyme at 100°C for 20min, and then centrifuging to obtain a supernatant, and then dialyzing the solution for 48h using a 10kDa dialysis bag, and then concentrating and freeze-drying the solution to obtain ulva polysaccharide; The preparation of spirulina polysaccharide comprises the following steps: weighing spirulina powder, and uniformly mixing the spirulina powder with water at a mass volume ratio of 1g:40mL; then, ultrasonically extracting for 120min at 60°C, 50kHz, and 200W, and then centrifuging at 5000rpm for 10min to obtain a supernatant, adding a 95% ethanol solution by volume to the obtained supernatant at a volume ratio of 1:4, precipitating overnight at 4°C, and then centrifuging to obtain a precipitate, and drying at 50°C to obtain a crude extract I; dissolving the extract I in water at a mass volume ratio of 1g:100mL, adding a protease to the solution to make the enzyme activity 250U / mL, and performing enzymolysis at 40°C for 150min, then inactivating the enzyme at 100°C for 20min, centrifuging to obtain a supernatant, and then dialyzing for 48h using a 10kDa dialysis bag, and then concentrating and freeze-drying to obtain spirulina polysaccharide.

2. The culture medium breeding method of Bletilla striata according to claim 1, characterized in that: In step S2, the culture conditions are: temperature 22-25°C, humidity 60-80%, and light intensity 4000-6000 lux.

3. The culture medium breeding method of Bletilla striata according to claim 1, characterized in that: The amino acid is selected from one of glycine, lysine, arginine and histidine or a mixture of two or more thereof.

4. The culture medium breeding method of Bletilla striata according to claim 1, characterized in that: In step S3, the culture conditions are: temperature 22-25° C., humidity 60-80%, and light intensity 4000-6000 lux.

5. The culture medium breeding method of Bletilla striata according to claim 1, characterized in that: In step S4, the culture soil is composed of 100-150 parts of garden soil, 10-30 parts of wood ash, 10-40 parts of rapeseed cake, 5-10 parts of humus soil and 20-40 parts of soybean residue powder in parts by weight.

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