Method for quickly transferring pleione bulbocodioides after sterile sowing of Yunnan pleione bulbocodioides

Abstract

The invention provides a method for rapidly transferring pleione bulbocodioides after sterile sowing, and relates to the technical field of tissue culture, the method comprises the following steps: providing pleione bulbocodioides pods; providing a primary culture medium; carrying out pod sowing and primary culture; subculture transfer: pouring the primary culture medium full of the protocorms into a device containing a subculture medium, and spreading the primary culture medium. According to the method, the transfer of the subculture is completed by pouring and sub-packaging, so that the waste of a primary culture medium is avoided, the time required by the transfer of the subculture tissue material is shortened, the time cost of seedling production is effectively reduced, and meanwhile, the yellowing or differentiation blocking of protocorm caused by deterioration of a microenvironment in a tissue culture bottle due to an overlong transfer period is avoided; the subsequent seedling production quality is improved; compared with a traditional mode of transferring by using tweezers, the method has the advantages that the time of exposing the tissue material in an environment outside a bottle is greatly shortened, and the pollution rate of the tissue material is effectively reduced.

Description

Technical Field

[0001] The present application relates to the field of tissue culture technology, and in particular to a method for rapid transplantation of Yunnan Orchidaceae after aseptic sowing. Background Art

[0002] Yunnan single garlic orchid ( Pleione yunnanensis Pleione, a member of the Orchidaceae family, belongs to the genus Pleione. Its pseudobulbs hold significant medicinal value and are one of the basal plants of the traditional Chinese medicine Psoralea corylifolia. They possess properties such as clearing heat and detoxifying, resolving phlegm and dispersing stagnation, and fighting cancer. It is primarily distributed in Yunnan, Sichuan, Guizhou, and Tibet, China, growing in humid mountainous areas at altitudes between 1,500 and 3,000 meters. In recent years, market demand for Pleione in Yunnan has been increasing, but its natural seed germination rate is low, leading to slow plant growth. Therefore, tissue culture, capable of rapidly obtaining large numbers of intact regenerated plants, has become a crucial technological solution and a major means of alleviating resource and environmental pressures. Recent reports on Pleione tissue culture have primarily focused on optimizing culture medium formulations, with limited coverage of optimized procedures. Traditional subculture often relies on manual transfer using forceps. This method is labor-intensive, time-consuming, and prone to tissue damage. Prolonged exposure of tissue material outside the flask increases contamination, hindering seedling production. Summary of the Invention

[0003] In view of this, the purpose of this application is to provide a method for rapid transfer of Yunnan Orchid after sterile sowing, in order to solve the problem that the existing manual transfer method using tweezers consumes a lot of manpower, material resources and time, and is also prone to tissue damage and contamination. The technical solution adopted by this application to solve the above technical problems is: An embodiment of the present invention provides a method for rapid transfer of Yunnan Orchid after aseptic sowing, comprising the following steps: providing Yunnan Orchid fruit pods; Provide primary culture medium; Fruit pod sowing and primary culture; Subculture transfer: Pour the primary culture medium covered with protocorms into a device containing subculture medium and spread it out.

[0004] In some embodiments, the primary culture medium comprises solid raw materials, agar powder and liquid raw materials, wherein the solid raw materials include acid hydrolyzed casein, activated carbon powder and white sugar; the liquid raw materials include macroelement mother liquor, trace element mother liquor, organic matter mother liquor, iron salt mother liquor, 6-BA mother liquor, NAA mother liquor and tap water; and / or The primary culture medium is in a soft gel state.

[0005] In some embodiments, M liters of primary culture medium includes M liters of tap water and M times of other raw materials, wherein the other raw materials include 0.8g~1.2g of acid-hydrolyzed casein, 1g~1.5g of activated carbon powder, 20g~30g of white sugar, 2.5g~3.5g of agar powder, 25ml~28ml of macroelement mother liquor, 2.5ml~3.0ml of trace element mother liquor, 4ml~5ml of organic mother liquor, 5ml~6ml of iron salt mother liquor, 2.5ml~5ml of 6-BA mother liquor, and 10ml~12ml of NAA mother liquor; wherein M is a positive rational number, the concentration of 6-BA mother liquor is 0.1mg / ml, and the concentration of NAA mother liquor is 0.1mg / ml.

[0006] In some embodiments, the fruit pods are sterilized, the sterilized fruit pods are cut off with a cooled scalpel, and then split open along the ridge to form 3 to 4 petals, revealing white loose embryos.

[0007] In some embodiments, the white loose embryos are evenly sown on the surface of the primary culture medium, and then cultured in the dark in a culture room at a temperature of T1 for x days. The Yunnan garlic orchid germinates and forms a yellow-green protocorm, and then enters the light culture stage with a culture temperature of T2, a light intensity of E1, and a light time of t1.

[0008] In some embodiments, T1 is 23°C to 26°C; and / or x days is 15 to 25 days; and / or T2 is 22°C to 25°C; and / or E1 is 1200x~1500x; and / or t1 is 8 hours / day to 10 hours / day.

[0009] In some embodiments, the protocorm covered with primary culture medium is a protocorm in the protocorm formation stage to the protocorm bud elongation stage; and / or The primary culture medium covered with the protocorm is shaken thoroughly to mix the primary culture medium and the protocorm thoroughly. At this time, the primary culture medium is in a slurry state. The primary culture medium covered with the protocorm is then poured into the device containing the secondary culture medium.

[0010] In some embodiments, 1 ml to 3 ml of primary culture medium covering the protocorm is poured into each device; and / or The volume content of the subculture culture medium in the device is 14%~18%.

[0011] In some embodiments, the poured primary culture medium covered with protocorms is spread out and then light cultured, with a light intensity of E2, a culture temperature of T3, and a light time of t2.

[0012] In some embodiments, E2 is 1200x to 1800x; and / or T3 is 25°C to 28°C; and / or t2 is 8 hours / day to 12 hours / day.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The present application adopts the method of pouring and sub-packaging to complete the transfer of subculture, which not only avoids the waste of primary culture medium and saves economic costs, but also shortens the time required for the transfer of subculture tissue materials, effectively reducing the time cost of seedling production. At the same time, it avoids the deterioration of the microenvironment in the tissue culture bottle caused by too long a transfer cycle, resulting in yellowing or differentiation obstruction of the protocorm, thereby improving the quality of subsequent seedling production; pouring the mixed protocorm culture medium to complete the transfer of subculture is different from the traditional method of using tweezers to transfer, which greatly shortens the time that the tissue material is exposed to the environment outside the bottle and effectively reduces the contamination rate of the tissue material. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following briefly introduces the drawings of the embodiments. Obviously, the drawings described below only relate to some embodiments of the present application and are not intended to limit the present application. Figure 1 The present invention is a flow chart of a method for rapid transfer of Yunnan Orchid after aseptic sowing; Figure 2 This is a picture of the embryo germination of the primary culture; Figure 3 This is a diagram of the bud point elongation of the primary cultured protocorm; Figure 4 This is a diagram of the primary culture medium for shaking and mixing protocorms; Figure 5 This is a picture of subculture and seedling formation. DETAILED DESCRIPTION

[0015] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of this application. In addition, it should be understood that the specific embodiments described herein are only used to illustrate and explain the present application and are not used to limit the present application.

[0016] In this application, unless otherwise specified, directional words such as "upper" and "lower" generally refer to the upper and lower parts of the device in actual use or working state; in addition, in the description of this application, the term "including" means "including but not limited to".

[0017] In this application, "and / or" describes the association relationship between associated objects, indicating that three relationships can exist. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0018] In this application, "at least one" means one or more, and "plurality" means two or more. "One or more," "at least one of the following," or similar expressions refer to any combination of these items, including any combination of single or plural items. For example, "at least one of a, b, or c" or "at least one of a, b and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can each be single or plural.

[0019] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be understood as a hard limitation on the scope of the present application; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numbers within the range. For example, the description of a range from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which applies regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0020] First, see Figure 1 The embodiment of the present invention provides a method for rapid transfer of Yunnan Orchid after aseptic sowing, comprising the following steps: S01. Provide Yunnan orchid fruit pods; S02, providing primary culture medium; S03, pod sowing and primary culture; S04. Subculture transfer: Pour the primary culture medium covered with the protocorm into a device containing the subculture culture medium and spread it out.

[0021] The present application adopts the method of pouring and sub-packaging to complete the transfer of subculture, which not only avoids the waste of primary culture medium and saves economic costs, but also shortens the time required for the transfer of subculture tissue materials, effectively reducing the time cost of seedling production. At the same time, it avoids the deterioration of the microenvironment in the tissue culture bottle caused by too long a transfer cycle, resulting in yellowing or differentiation obstruction of the protocorm, thereby improving the quality of subsequent seedling production; pouring the mixed protocorm culture medium to complete the transfer of subculture is different from the traditional method of using tweezers to transfer, which greatly shortens the time that the tissue material is exposed to the environment outside the bottle and effectively reduces the contamination rate of the tissue material.

[0022] In the S01: In some embodiments, the pods are collected when the embryos are white and fluffy.

[0023] For example, artificial assisted pollination is carried out during the flowering period of Yunnan Orchid from April to May, and the fruit pods are collected after 5 months of development when the embryo is in a white and loose state.

[0024] It is understandable that the pods are collected when the embryos are white and loose. During this period, the appropriate time for picking the pods can be determined through regular sampling and inspection to avoid the embryos being too young and tender, affecting the subsequent tissue culture and sterile sowing, and also to avoid the embryos being too mature, affecting the embryo germination rate.

[0025] In the S02: In some embodiments, the primary culture medium includes solid raw materials, agar powder and liquid raw materials, wherein the solid raw materials include acid-hydrolyzed casein, activated carbon powder and white sugar; the liquid raw materials include macroelement mother liquor, trace element mother liquor, organic matter mother liquor, iron salt mother liquor, 6-BA mother liquor, NAA mother liquor and tap water.

[0026] Furthermore, M liters of primary culture medium includes M liters of tap water and M times of other raw materials, wherein the other raw materials include 0.8g~1.2g of acid-hydrolyzed casein, 1g~1.5g of activated carbon powder, 20g~30g of white sugar, 2.5g~3.5g of agar powder, 25ml~28ml of macroelement mother liquor, 2.5ml~3.0ml of trace element mother liquor, 4ml~5ml of organic matter mother liquor, 5ml~6ml of iron salt mother liquor, 2.5ml~5ml of 6-BA mother liquor, and 10ml~12ml of NAA mother liquor; wherein M is a positive rational number.

[0027] Furthermore, M liters of primary culture medium includes M liters of tap water and M times of other raw materials, wherein the other raw materials include 0.8g-1.2g of acid hydrolyzed casein, for example, 0.8g, 0.9g, 1g, 1.1g, 1.2g, etc.; 1g-1.5g of activated carbon powder, for example, 1g, 1.1g, 1.2g, 1.3g, 1.4g, 1.5g, etc.; 20g-30g of white sugar, for example, 20g, 22g, 25g, 26g, 28g, 30g, etc.; 2.5g-3.5g of agar powder, for example, 2.5g, 2.6g, 2.8g, 3g, 3.2g, 3.3g, 3.5g, etc.; 25ml-28ml of macronutrient solution, for example, 25ml, 26ml, 27ml, 28ml, etc.; 2. 5ml~3.0ml, for example, it can be 2.5ml, 2.6ml, 2.7ml, 2.8ml, 2.9ml, 3ml, etc.; organic mother liquor can be 4ml~5ml, for example, it can be 4ml, 4.2ml, 4.5ml, 4.6ml, 4.8ml, 5ml, etc.; iron salt mother liquor can be 5ml~6ml, for example, it can be 5ml, 5.1ml, 5.2ml, 5.5ml, 5.8ml, 6ml, etc.; 6-BA mother liquor can be 2.5ml~5ml, for example, it can be 2.5ml, 2.8ml, 3ml, 3.5ml, 4ml, 4.2ml, 4.8ml, 5ml, etc.; NAA mother liquor can be 10ml~12ml, for example, it can be 10ml, 11ml, 11.5ml, 11.8ml, 12ml, etc.; wherein M is a positive rational number.

[0028] Furthermore, the preparation method of M liters of primary culture medium includes: boiling M liters of tap water, then pouring M times of 2.5g~3.5g of agar powder into the boiling water, stirring to fully dissolve, and then adding M times of solid raw materials, the solid raw materials include 0.8g~1.2g of acid-hydrolyzed casein, 1g~1.5g of activated carbon powder, and 20g~30g of white sugar, stirring to dissolve, and when the tap water starts to boil again, quickly pouring M times of liquid raw materials, the liquid raw materials include 25ml~28ml of macroelement mother liquor, 2.5ml~3.0ml of trace element mother liquor, 4ml~5ml of organic mother liquor, 5ml~6ml of iron salt mother liquor, 2.5ml~5ml of 6-BA mother liquor, and 10ml~12ml of NAA mother liquor to obtain a semi-finished product, and after slight stirring, adjusting the pH of the semi-finished product to 5.8~6.0 with dilute hydrochloric acid to obtain the primary culture medium.

[0029] Furthermore, the concentration of the 6-BA stock solution was 0.1 mg / ml, and the concentration of the NAA stock solution was 0.1 mg / ml.

[0030] Furthermore, to prepare 1L of macronutrient solution, weigh 33000mg of analytically pure NH4NO3, 38000mg of KNO3, 8800mg of CaCl2·2H2O, 7400mg of MgSO4·7H2O, and 3400mg of KH2PO4, with the remainder being distilled water. To prepare 1 L of trace element mother solution, weigh 166 mg of analytically pure crystalline KI, 1240 mg of H3BO3, 4460 mg of MnSO4·4H2O, 1720 mg of ZnSO4·7H2O, 50 mg of Na2MoO4·2H2O, 5 mg of CuSO4·5H2O, and 5 mg of CoCl2·6H2O, with the balance being distilled water. To prepare 1 L of iron salt mother solution, weigh 5560 mg of analytically pure FeSO4·7H2O, 7460 mg of Na2-EDTA, and the balance is distilled water; To prepare 1 L of organic stock solution, you need to weigh 20,000 mg of analytically pure inositol, 100 mg of niacin (VB5), 100 mg of pyridoxine hydrochloride (vitamin B6), 20 mg of thiamine hydrochloride (vitamin B1), and 400 mg of glycine, with the balance being distilled water.

[0031] For example, 1L of purified tap water is boiled, and then 3.0g of agar powder is poured into the boiling water, stirred to fully dissolve, and then the solid mixed raw materials are added, which include 1g of acid-hydrolyzed casein, 1g of activated carbon powder, and 30g of white sugar. Stir to dissolve. When the tap water starts to boil again, the liquid raw materials are quickly poured in. The liquid raw materials include 25ml of macroelement mother liquor, 2.5ml of trace element mother liquor, 5ml each of organic mother liquor and iron salt mother liquor, 2.5ml of 6-BA mother liquor with a concentration of 0.1mg / ml, and 10ml of NAA mother liquor with a concentration of 0.1mg / ml. After gently stirring several times, the pH of the culture medium is adjusted to 5.8-6.0 with 5% dilute hydrochloric acid to obtain the primary culture medium.

[0032] It can be understood that 6-BA mother liquor is 6-benzylaminopurine solution; NAA mother liquor is α-naphthaleneacetic acid solution.

[0033] In some embodiments, the method for preparing the primary culture medium comprises the following steps: S021. Bring 1 L of purified tap water to a boil, then pour 3.0 g of agar powder into the boiling water and stir to dissolve thoroughly. Then, add the solid mixed raw materials, which include 1 g of acid-hydrolyzed casein, 1 g of activated carbon powder, and 30 g of white sugar. Stir to dissolve. When the tap water begins to boil again, quickly pour in the liquid raw materials, which include 25 ml of a large element mother liquor, 2.5 ml of a trace element mother liquor, 5 ml each of an organic matter mother liquor and an iron salt mother liquor, 2.5 ml of a 6-BA mother liquor with a concentration of 0.1 mg / ml, and 10 ml of a NAA mother liquor with a concentration of 0.1 mg / ml. After gently stirring several times, adjust the pH of the culture medium to 5.8-6.0 with 5% dilute hydrochloric acid to obtain the primary culture medium, which is then canned and bottled. S022. Each bottle is filled with about 50ml~60ml of culture medium. After filling, sterilize it at 121℃ for 25 minutes and cool it down after sterilization.

[0034] Furthermore, the primary culture medium is in a soft gel state.

[0035] It can be understood that the sterilized culture medium should be placed in a clean and ventilated environment in time to facilitate rapid cooling of the culture medium. At this time, the solidified state of the culture medium is that the culture medium can be slightly shaken when the bottle is shaken, and the culture medium is in a flowing slurry state when the bottle is shaken vigorously. The culture medium at this time is the required culture medium.

[0036] In said S03: See also Figure 2 In some embodiments, the fruit pods are sterilized, the sterilized fruit pods are cut off with a cooled scalpel, and then cut open along the ridge to form 3 to 4 petals, revealing white loose embryos.

[0037] Furthermore, the white loose embryos are evenly spread on the surface of the primary culture medium, and then cultured in the dark in a culture room at a temperature of T1 for x days. The Yunnan garlic orchid germinates and forms yellow-green protocorms, which then enter the light culture stage with a culture temperature of T2, a light intensity of E1, and a light time of t1.

[0038] Furthermore, T1 is 23°C to 26°C, for example, it can be 23°C, 24°C, 25°C, 26°C, etc.

[0039] x days is 15 to 25 days, for example, it can be 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, etc.

[0040] T2 is 22°C to 25°C, for example, it can be 22°C, 23°C, 24°C, 25°C, etc.

[0041] E1 is 1200x~1500x, for example, it can be 1200x, 1300x, 1400x, 1500x, etc.

[0042] t1 is 8 hours / day to 10 hours / day, for example, it can be 8 hours / day, 9 hours / day, 10 hours / day, etc.

[0043] In said S04: In some embodiments, the transfer of subculture is as follows: the primary culture medium covered with the protocorm is poured into a bottle containing the subculture medium, the bottle cap is closed, and the bottle is gently shaken to spread the poured primary culture medium covered with the protocorm.

[0044] See also Figure 3 Furthermore, the protocorm covered with the primary culture medium is the protocorm from the protocorm formation stage to the protocorm bud elongation stage.

[0045] Furthermore, 1 ml to 3 ml of the primary culture medium filled with protocorms is poured into each device, for example, 1 ml, 1.2 ml, 1.5 ml, 1.8 ml, 2 ml, 2.2 ml, 2.3 ml, 2.5 ml, 2.6 ml, 2.8 ml, 2.9 ml, 3 ml, etc.

[0046] See also Figure 4 Further, the primary culture medium covered with the protocorm is fully shaken to fully mix the primary culture medium and the protocorm. At this time, the primary culture medium is in a slurry state, and then the primary culture medium covered with the protocorm is poured into the device containing the secondary culture medium.

[0047] Furthermore, the volume content of the secondary culture medium in the device is 14% to 18%, for example, 14%, 15%, 16%, 17%, 18%, etc.

[0048] Illustratively, the specification of the empty bottle is 350 ml, and the amount of the subculture culture medium is 50 ml to 60 ml, for example, it can be 50 ml, 52 ml, 55 ml, 56 ml, 58 ml, 60 ml, etc.

[0049] See also Figure 5 Furthermore, the primary culture medium filled with protocorms is spread out and then light cultured, with a light intensity of E2, a culture temperature of T3, and a light time of t2.

[0050] Furthermore, E2 is 1200x~1800x, for example, it can be 1200x, 1300x, 1400x, 1500x, 1600x, 1700x, 1800x, etc.

[0051] T3 is 25°C to 28°C, for example, it can be 25°C, 26°C, 27°C, 28°C, etc.

[0052] t2 is 8 hours / day to 12 hours / day, for example, it can be 8 hours / day, 9 hours / day, 10 hours / day, 11 hours / day, 12 hours / day, etc.

[0053] It will be appreciated that the secondary culture medium is conventional in the art and can be obtained commercially.

[0054] Test Example 1 In order to explore the appropriate amount of agar powder added to the primary culture medium, five addition amount gradients were set up, and the results are shown in Table 1.

[0055] Table 1 Comparison of the effects of different agar powder additions on embryo germination

[0056] The results showed that when the amount of agar powder added was in the range of 0-1.5g / L, the primary culture medium was in liquid state, the embryo germination rate of Yunnan Orchid was low, and the protocorm sank to the bottom of the bottle, the color was white, and the bud differentiation was blocked; in the range of 2.5-3.5g / L, the embryo germination rate was relatively high, the germinated protocorm was on the surface of the culture medium, was yellow-green, and the bud differentiation was normal. The amount of agar powder added in this range met the operational requirements. When the amount of agar powder added was 4.5g / L, the embryo germination rate was high and the protocorm differentiation was normal, but the culture medium was hard and not easy to shake, which was not conducive to the next step of subculture. Considering the embryo germination rate and the hardness of the culture medium, the amount of agar powder added in the range of 2.5-3.5g / L met the operational requirements.

[0057] Test Example 2 The comparison of the effects of dumping and grafting of protocorms at different differentiation stages is shown in Table 2.

[0058] Table 2 Comparison of the effect of dumping and grafting of protocorms at different differentiation stages

[0059] The results showed that the optimal time for transferring Orchidaceae to subculture vials is from protocorm formation to protocorm bud elongation. The contamination rate is relatively low, and the protocorms are easily dispersed after transfer. During the bud elongation stage, the white root hairs formed by the protocorms begin to gradually become dense, but the protocorms tend to clump after transfer vials, resulting in poor transfer efficiency. During the leaf differentiation stage, protocorms transferred vials often clump, making the tipping transfer method unsuitable at this stage. Considering the contamination rate and the degree of dispersion of the transferred protocorms, the optimal time for transferring Orchidaceae to subculture vials is from protocorm formation to protocorm bud elongation.

[0060] Test Example 3 The same experimental operator used two subculture transfer methods respectively, and compared the transfer efficiency and contamination rate after transfer and bottle division, as shown in Table 3.

[0061] Table 3 Comparison of the effects of traditional tweezers cluster transfer and pouring bottle transfer

[0062] The results showed that the transfer method of pouring the bottle was 2.57 times more efficient than the traditional transfer method of dividing the clusters with tweezers, and the contamination rate was 41.67% lower than the traditional transfer method. The transfer method of pouring the bottle is superior to the traditional transfer method.

[0063] The basic concepts have been described above. It will be apparent to those skilled in the art that the detailed disclosure above is merely illustrative and does not limit the present application. Although not explicitly stated herein, those skilled in the art may make various modifications, improvements, and amendments to the present application. Such modifications, improvements, and amendments are suggested in the present application and remain within the spirit and scope of the exemplary embodiments of the present application.

[0064] At the same time, this application uses specific terms to describe the embodiments of this application. For example, "one embodiment," "an embodiment," and / or "some embodiments" refer to a certain feature, structure, or characteristic related to at least one embodiment of this application. Therefore, it should be emphasized and noted that "one embodiment," "an embodiment," or "an alternative embodiment" mentioned twice or multiple times in different locations in this specification does not necessarily refer to the same embodiment. In addition, certain features, structures, or characteristics in one or more embodiments of this application may be appropriately combined.

Claims

1. A method for rapid transfer after aseptic sowing of Yunnan Orchid, characterized in that: The steps include: Provide Yunnan Du Suanlan fruit pods; Provide primary culture medium; Fruit pod sowing and primary culture; Subculture transfer: Pour the primary culture medium covered with protocorms into a device containing subculture medium and spread it out.

2. The method for rapid transfer after aseptic sowing of Yunnan garlic orchid according to claim 1, wherein The primary culture medium comprises solid raw materials, agar powder and liquid raw materials, wherein the solid raw materials include acid hydrolyzed casein, activated carbon powder and white sugar; the liquid raw materials include macroelement mother liquor, trace element mother liquor, organic matter mother liquor, iron salt mother liquor, 6-BA mother liquor, NAA mother liquor and tap water; and / or The primary culture medium is in a soft gel state.

3. The method for rapid transfer after aseptic sowing of Yunnan garlic orchid according to claim 2, wherein M liters of primary culture medium includes M liters of tap water and M times of other raw materials, wherein the other raw materials include 0.8g~1.2g of acid-hydrolyzed casein, 1g~1.5g of activated carbon powder, 20g~30g of white sugar, 2.5g~3.5g of agar powder, 25ml~28ml of macroelement mother liquor, 2.5ml~3.0ml of trace element mother liquor, 4ml~5ml of organic matter mother liquor, 5ml~6ml of iron salt mother liquor, 2.5ml~5ml of 6-BA mother liquor, and 10ml~12ml of NAA mother liquor; wherein M is a positive rational number, the concentration of 6-BA mother liquor is 0.1mg / ml, and the concentration of NAA mother liquor is 0.1mg / ml.

4. The method for rapid transfer after aseptic sowing of Yunnan garlic orchid according to claim 1, wherein Disinfect the pods, cut off the heads and tails of the sterilized pods with a cooled scalpel, and then split them into 3 to 4 petals along the ridge to reveal the white, loose embryo.

5. The method for rapid transfer after aseptic sowing of Yunnan garlic orchid according to claim 4, wherein The loose white embryos were evenly spread on the surface of the primary culture medium, and then cultured in the dark in a culture room at a temperature of T1 for x days. The Yunnan garlic orchid germinated and formed yellow-green protocorms, and then entered the light culture stage with a culture temperature of T2, a light intensity of E1, and a light time of t1.

6. The method for rapid transfer after aseptic sowing of Yunnan garlic orchid according to claim 5, wherein T1 is 23°C to 26°C; and / or x days is 15 to 25 days; and / or T2 is 22°C to 25°C; and / or E1 is 1200x~1500x; and / or t1 is 8 hours / day to 10 hours / day.

7. The method for rapid transfer after aseptic sowing of Yunnan garlic orchid according to claim 1, wherein The protocorm covered with the primary culture medium is a protocorm in the protocorm formation stage to the protocorm bud elongation stage; and / or The primary culture medium covered with the protocorm is shaken thoroughly to mix the primary culture medium and the protocorm thoroughly. At this time, the primary culture medium is in a slurry state. The primary culture medium covered with the protocorm is then poured into the device containing the secondary culture medium.

8. The method for rapid transfer after aseptic sowing of Yunnan garlic orchid according to claim 1, wherein Pour 1ml~3ml of primary culture medium filled with protocorms into each device; and / or The volume content of the subculture culture medium in the device is 14%~18%.

9. The method for rapid transfer after aseptic sowing of Yunnan garlic orchid according to claim 1, wherein The primary culture medium filled with protocorms is spread out and then light culture is carried out, with a light intensity of E2, a culture temperature of T3, and a light time of t2.

10. The method for rapid transfer after aseptic sowing of Yunnan Orchidaceae according to claim 9, characterized in that, E2 is 1200x~1800x; and / or T3 is 25°C to 28°C; and / or t2 is 8 hours / day to 12 hours / day.

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