Method for culturing anoectochilus formosanus protocorm-like bodies

Abstract

The invention provides a method for culturing anoectochilus formosanus protocorm-like bodies, and particularly selects Jinkang No.1 as a culture variety. By optimizing the inoculum size and the culture time, efficient proliferation of the protocorm-like body and accumulation of high medicinal components are achieved, when the protocorm-like body is cultured for 30 days with the inoculum size of 60 g / L, the proliferation coefficient reaches up to 1.32, and the content of kinsenoside is remarkably increased to 8.51 + / -0.56 mg / g. When the inoculation amount is lower than 60g / L, the content of kinsenoside is relatively low; and when the culture time exceeds 60g / L, although the content of the kinsenoside is increased, the increase speed is slower, and after the culture time is prolonged, the content of the kinsenoside is reduced on the contrary. Therefore, the culture condition for 30 days at the inoculation amount of 60g / L is the final selected culture condition, so that not only is the efficient proliferation of the protocorm-like body ensured, but also the high content of the kinsenoside is ensured, more lateral buds are generated, and the propagation is facilitated. The method provides powerful support for large-scale production and medicinal development of the anoectochilus formosanus, and the proliferation efficiency and medicinal value of the anoectochilus formosanus are remarkably improved.

Description

Technical Field

[0001] The invention belongs to the technical field of roxburghii, and particularly relates to a method for culturing roxburghii-like protocorms. Background Art

[0002] Protocorm-like bodies (PLBs) of Anoectochilus roxburghii are a special structure in tissue culture of orchids. They are induced by plant tissue culture technology under sterile conditions, similar to the protocorms formed when seeds germinate under natural conditions. Protocorm-like bodies are morphologically similar to protocorms, but they are formed by cell division and differentiation in plant tissue culture.

[0003] However, existing studies have shown that the induction rate of primary protocorms of A. roxburghii is relatively low and the growth rate is also slow. For example, a study pointed out that when stem segments were used as explants, the induction rate of primary protocorms of A. roxburghii was only 66%. In addition, the induction rate of secondary protocorms of A. roxburghii is also low and the yield is not high, which limits its application in rapid proliferation and production of medicinal ingredients.

[0004] More importantly, most of the protocorms of A. roxburghii contain low levels of anoectin, which affects its value as a medicinal plant. Anoectin is an important bioactive ingredient in A. roxburghii with multiple pharmacological effects, which leads to low efficiency in the development of medicinal value and commercial production of A. roxburghii protocorms. Summary of the invention

[0005] The invention provides a method for culturing anoectochilus protocorms and an induction culture medium, aiming to solve the technical problem of low efficiency of the method for obtaining anoectochilus glycoside in the prior art.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides a method for culturing a roxburghii-like protocorm, comprising:

[0008] S1. Cultivation of primary protocorms of Anoectochilus roxburghii: Take a 1-1.5 cm stem segment of Jinkang No. 1 Anoectochilus roxburghii seedling, inoculate it into an induction medium, and obtain primary protocorms of Anoectochilus roxburghii in 28-32 days under the conditions of 23-27°C and 16h / 8h light-dark cycle;

[0009] S2. Cultivation of secondary protocorms of Anoectochilus roxburghii: taking the buds of the primary protocorms of Anoectochilus roxburghii, cutting them into 2-4 mm slices, inoculating them into an induction medium, and obtaining secondary protocorms of Anoectochilus roxburghii for 58-62 days under the conditions of 23-27° C. and 16h / 8h light-dark cycle;

[0010] S3. Proliferation of secondary protocorms: inoculating the secondary protocorms of Anoectochilus roxburghii into a bioreactor at an inoculation rate of 55-65 g / L, inducing with an induction medium, and culturing for 38-42 days under aeration of 550-650 mL / min and dim light conditions to obtain protocorms of Anoectochilus roxburghii;

[0011] The induction culture medium comprises MS culture medium, 2.5-3.5 mg / L 6-benzyladenine, 0.4-0.6 mg / L 1-naphthylacetic acid, 0.7-0.9 mg / L zeatin and 0.1-0.3 mg / L 2,4-dichlorophenoxyacetic acid.

[0012] In a preferred embodiment, the induction medium comprises MS medium, 3.2 mg / L 6-benzyladenine, 0.4 mg / L 1-naphthylacetic acid, 0.75 mg / L zeatin and 0.12 mg / L 2,4-dichlorophenoxyacetic acid.

[0013] In a preferred embodiment, the induction medium comprises MS medium, 2.5 mg / L 6-benzyladenine, 0.6 mg / L 1-naphthylacetic acid, 0.9 mg / L zeatin and 0.3 mg / L 2,4-dichlorophenoxyacetic acid.

[0014] In a preferred embodiment, the induction medium comprises MS medium, 3.0 mg / L 6-benzyladenine, 0.5 mg / L 1-naphthylacetic acid, 0.8 mg / L zeatin and 0.2 mg / L 2,4-dichlorophenoxyacetic acid.

[0015] Based on the above scheme, experiments have proved that the use of a culture medium containing MS culture medium, 3.0 mg / L 6-benzyladenine, 0.5 mg / L 1-naphthaleneacetic acid, 0.8 mg / L zeatin and 0.2 mg / L 2,4-dichlorophenoxyacetic acid can significantly promote the growth of stem segments of Anoectochilus roxburghii tissue culture seedlings, so that protocorm-like bodies can be formed in just 23 days, with a high induction rate, and the protocorm-like body induction rate is as high as 95%, and the protocorm-like body length can reach 1.29 cm.

[0016] In a preferred embodiment, in step S1, the lighting condition of the photoperiod is 1500-20001ux.

[0017] In a preferred embodiment, in step S1, a 1.2 cm stem segment of a tissue cultured seedling of A. roxburghii is taken and inoculated into an induction medium to obtain a primary protocorm-like body of A. roxburghii under the conditions of 25° C. and a 16 h / 8 h light / dark cycle for 30 days.

[0018] A preferred solution is that in step S2, the buds of the primary protocorm-like bodies of Anoectochilus roxburghii are taken, cut into 3 mm thin slices, inoculated into an induction culture medium, and the secondary protocorm-like bodies of Anoectochilus roxburghii are obtained for 60 days under the conditions of 25° C. and 16h / 8h light / dark cycle.

[0019] According to a preferred embodiment, in step S3, the secondary protocorms of A. roxburghii are inoculated into a bioreactor at an inoculation amount of 55 g / L, induced with an induction medium, and cultured for 32 days under aeration of 620 mL / min and dim light conditions to obtain protocorms of A. roxburghii.

[0020] According to a preferred embodiment, in step S3, the secondary protocorms of A. roxburghii are inoculated into a bioreactor at an inoculation amount of 60 g / L, induced with an induction medium, and cultured for 30 days under aeration of 600 mL / min and dim light conditions to obtain protocorms of A. roxburghii.

[0021] According to a preferred embodiment, in step S3, the secondary protocorms of A. roxburghii are inoculated into a bioreactor at an inoculation amount of 60 g / L, induced with an induction medium, and cultured for 30 days under aeration of 600 mL / min and dim light conditions to obtain protocorms of A. roxburghii.

[0022] Based on the above scheme, under the conditions of ventilation of 500mL / min and dark light culture, the proliferation coefficient of bulb-like stems obtained by culturing for 30 days with an inoculum size of 60g / L was the highest.

[0023] The beneficial effects of the present invention are:

[0024] The present invention provides an efficient method for culturing protocorm-like bodies of Anoectochilus roxburghii, selects Jinkang No. 1 as a culture variety, and realizes a continuous culture process from primary protocorms of Anoectochilus roxburghii to secondary protocorms, and then to the proliferation of secondary protocorms-like bodies under an induction medium and optimized culture conditions. Among them, after culturing Jinkang No. 1 for 30 days with an inoculation amount of 60 g / L, compared with other varieties, the number of lateral buds formed by Jinkang No. 1 is the largest, and the large-scale formation of lateral buds helps to quickly increase the number of protocorms-like bodies, which not only improves the proliferation efficiency, but also ensures the high-quality proliferation of protocorms-like bodies, thereby improving the overall proliferation efficiency.

[0025] After 23 days of cultivation, the length of the primary protocorm of Jinkang No. 1 Anoectochilus was between 0.6 and 1.0 cm, and the induction rate exceeded 90%, showing a high induction efficiency. Furthermore, in the induction stage of the secondary protocorm-like bodies of Jinkang No. 1 Anoectochilus, Jinkang No. 1 not only grew rapidly, but also successfully induced protocorms-like bodies in just 25 days, and the induction rate also remained above 90%.

[0026] At the same time, compared with other varieties, Jinkang No. 1 golden thread vine induced milky white protocorms for horizontal expansion, and the induction rate of milky white protocorms was higher. It has the advantages of strong proliferation ability, fast germination speed and high accumulation of medicinal ingredients. The genetic traits of the milky white protocorms are stable, which is extremely beneficial for rapid proliferation and preservation of germplasm resources.

[0027] In addition, the present invention will be induced by Jinkang No. 1 golden thread lotus milky white secondary protocorm obtained by proliferation culture, the test results show that: the appropriate inoculum can significantly promote the proliferation of the bulb-like corms. The experiment found that when the inoculum was controlled at 60g / L, the proliferation coefficient and the content of aureoside increased the fastest, when the inoculum was lower than 60g / L, the proliferation coefficient and the content of aureoside were lower, when the inoculum was higher than 60g / L, the proliferation coefficient and aureoside increased slowly, therefore, the present invention controls the inoculum at 60g / L. In addition, at 30 days, the proliferation rate of the golden thread lotus protocorm was the fastest, and the proliferation coefficient increased significantly. After more than 30 days, the proliferation rate slowed down, and at 30 days, the aureoside content reached the highest. After more than 30 days, the aureoside content began to decline. Therefore, taking the proliferation coefficient and the content of anoectoside into consideration, the present invention controls the culture time to 30 days, at which time the proliferation coefficient is as high as 1.32, and the content of anoectoside is significantly increased to 8.51±0.56. This culture condition not only ensures the efficient proliferation of the bulb-like stems, but also ensures the high content of anoectoside, and this content level not only ensures the high medicinal value of anoectoside.

[0028] By means of the cultivation method of the present invention, a large amount of roxburghii protocorms with high roxburghii glycoside content can be quickly obtained, and roxburghii glycoside can be produced quickly and efficiently. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 These are photos of primary protocorms of Anoectochilus roxburghii induced from different provenances of Anoectochilus roxburghii in the experiment of the present invention.

[0031] Figure 2 These are photos of secondary protocorms of Anoectochilus roxburghii induced from different provenances of Anoectochilus roxburghii in the experiment of the present invention.

[0032] Figure 3 This is a photo of the secondary protocorms induced by Jinkang No. 1 golden thread vine.

[0033] Figure 4 This is a photo of the lateral buds of the protocorm of a single Jinkang No. 1 golden thread vine.

[0034] Figure 5 These are photos of the lateral buds of several Jinkang No. 1 golden thread vine protocorms. DETAILED DESCRIPTION

[0035] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0036] This embodiment provides a method for culturing a roxburghii-like protocorm, comprising:

[0037] S1. Cultivation of primary protocorms of Anoectochilus roxburghii: Take a 1-1.5 cm stem segment of Jinkang No. 1 Anoectochilus roxburghii seedling, inoculate it into an induction medium, and culture it for 28-32 days at 23-27° C. and 16h / 8h light-dark cycle to obtain primary protocorms of Anoectochilus roxburghii;

[0038] Specifically, a 1.2 cm stem segment of a tissue culture seedling of A. roxburghii was taken, inoculated into an induction medium, and cultured for 30 days at 25° C. and a 16 h / 8 h light / dark cycle to obtain a primary protocorm-like body of A. roxburghii, wherein the light condition of the photoperiod was 1500-2000 lux.

[0039] S2. Cultivation of secondary protocorms of Anoectochilus roxburghii: taking the buds of the primary protocorms of Anoectochilus roxburghii, cutting them into 2-4 mm slices, inoculating them into an induction medium, and obtaining secondary protocorms of Anoectochilus roxburghii for 58-62 days under the conditions of 23-27° C. and 16h / 8h light-dark cycle;

[0040] S3. Proliferation of secondary protocorms: inoculating the secondary protocorms of Anoectochilus roxburghii into a bioreactor at an inoculation amount of 55-65 g / L, inducing with an induction medium, and culturing for 28-42 days under aeration of 550-650 mL / min and dim light conditions to obtain protocorms of Anoectochilus roxburghii;

[0041] Specifically, the bioreactor can be a culture dish or a bioreactor for culturing protocorms of Anoectochilus provided by Patent No. ZL 202323404893.X, wherein the bioreactor is provided with a transparent outer cover outside a transparent culture tank, a part of the culture tank extends out of the outer cover, and a culture cavity is provided on the part of the culture tank located inside the outer cover; a temperature control component for controlling the temperature of the culture cavity is provided between the outer cover and the culture tank; an illuminator is provided outside the outer cover, and the light emitted by the illuminator is irradiated on the culture in the culture cavity. Cultivating protocorms of Anoectochilus by the bioreactor can reduce the cost of extracting components such as Anoectochilus sugar in the later stage.

[0042] The induction culture medium comprises MS culture medium, 2.5-3.5 mg / L 6-benzyladenine, 0.4-0.6 mg / L 1-naphthylacetic acid, 0.7-0.9 mg / L zeatin and 0.1-0.3 mg / L 2,4-dichlorophenoxyacetic acid.

[0043] Wherein, the induction culture medium comprises MS culture medium, 3.2 mg / L 6-benzyladenine, 0.4 mg / L 1-naphthylacetic acid, 0.75 mg / L zeatin and 0.12 mg / L 2,4-dichlorophenoxyacetic acid.

[0044] Alternatively, the induction medium comprises MS medium, 2.5 mg / L 6-benzyladenine, 0.6 mg / L 1-naphthylacetic acid, 0.9 mg / L zeatin and 0.3 mg / L 2,4-dichlorophenoxyacetic acid.

[0045] A more preferred solution: the induction culture medium includes MS culture medium, 3.0 mg / L 6-benzyladenine, 0.5 mg / L 1-naphthylacetic acid, 0.8 mg / L zeatin and 0.2 mg / L 2,4-dichlorophenoxyacetic acid.

[0046] Among them, in step S3, the secondary protocorms of Anoectochilus roxburghii are inoculated into a bioreactor at an inoculation amount of 55 g / L, induced with induction medium, and cultured for 32 days under aeration of 620 mL / min and dark light conditions to obtain protocorms of Anoectochilus roxburghii.

[0047] Alternatively, the secondary protocorm-like body of A. roxburghii is inoculated into a bioreactor at an inoculation amount of 65 g / L, induced with an induction medium, and cultured for 28 days under aeration of 550 mL / min and dim light conditions to obtain the protocorm-like body of A. roxburghii.

[0048] Among them, a better solution is: inoculate the secondary protocorms of Anoectochilus roxburghii into a bioreactor at an inoculation rate of 60 g / L, induce with induction medium, and culture for 30 days under ventilation volume of 600 mL / min and dark light conditions to obtain the protocorms of Anoectochilus roxburghii.

[0049] The present invention will be further explained below in conjunction with experiments:

[0050] 1. Cultivation of primary protocorms of Anoectochilus roxburghii

[0051] 1.1 Test methods

[0052] Taking tissue culture seedlings of five varieties of Anoectochilus roxburghii, namely, ZY1 (Jinkang No. 1), ZY2 (Taiwanese silver thread orchid), ZY3 (Wuping Anoectochilus roxburghii), ZY4 (large round-leaf Anoectochilus roxburghii), and ZY5 (Ganzhou wild Anoectochilus roxburghii), as test materials, an induction medium (MS+3mg / L6-BA+0.5mg / L NAA+0.8mg / L ZT+0.2mg / L2,4-D) provided by the present invention was used to carry out an induction test of solid protocorms.

[0053] First, the tissue culture seedlings of Anoectochilus roxburghii were taken out from the tissue culture bottle, the roots and leaves were removed, and then the stems were cut into 1-1.5 cm segments as explants. 20 explants were inoculated on each dish of culture medium, and repeated 7 times to ensure the reliability and repeatability of the test results. Protocorm-like body induction was carried out under the conditions of 25±2℃, 16h / 8h light-dark cycle (light intensity of 1500-2000lux). The time required for the appearance of protocorms was recorded, and the induction rate of protocorms-like bodies and their length were calculated 30 days after inoculation.

[0054] The calculation formula of the induction rate of protocorm-like bodies is: the number of explants containing protocorm-like bodies / the number of explants inoculated with the culture medium×100%.

[0055] 1.2 Test results

[0056] The induction rates of protocorms of the three varieties ZY1, ZY3, and ZY5 are all above 90%, among which ZY5 has the fastest growth rate, followed by ZY3, indicating that these three varieties are suitable for producing protocorms. The induction rates of ZY2 and ZY4 are relatively low, about 80% and 70% respectively, and the growth rate of ZY4 is the slowest. Subsequent experiments will continue to carry out repeated tests on the induction rate to further verify these results. The induction of primary protocorms of each variety is detailed in Table 1, Figure 1 and Figure 2 .

[0057] Table 1 Induction of primary protocorms of 15 varieties of Anoectochilus roxburghii (n=140)

[0058] variety Time of appearance of protocorm-like bodies Length of protocorm Protocorm-like body induction rate (%) ZY1 25.8±2.86 0.76±0.29 95±7.64 ZY2 24.6±3.97 0.98±0.37 80.71±9.76 ZY3 24.6±3.65 1.04±0.31 94.29±5.35 ZY4 28±1.58 0.76±0.32 71±6.52 ZY5 23±3.16 1.29±0.33 95±6.12

[0059] 2. Cultivation of secondary protocorms of Anoectochilus roxburghii

[0060] 2.1 Test methods

[0061] Based on the results of primary protocorm culture of Anoectochilus roxburghii, the primary buds of ZY1, ZY2, ZY3, ZY4, and ZY55 varieties were used as raw materials, and the induction medium MS+3mg / L6-BA+0.5mg / LNAA+0.8mg / L ZT+0.2mg / L2,4-D was used as the induction medium to induce the secondary protocorms of Anoectochilus roxburghii. The stem segments of Anoectochilus roxburghii after 30 days of culture were taken out, and the buds were cut into 2-4mm slices and inoculated in the optimized medium. 20 slices were inoculated per dish, and the time of induction of secondary protocorms was recorded. After 60 days of culture at 25±2℃ and dark light conditions, the growth morphology of secondary protocorms of different varieties was recorded to determine the best variety suitable for secondary protocorm culture.

[0062] 2.2 Test results

[0063] Refer to Table 2. According to the test results, the induction rates of protocorms of ZY1, ZY3, and ZY5 are all above 90%, with ZY1 having the highest induction rate and ZY4 having the lowest. The appearance time of secondary protocorms of ZY1, ZY2, ZY3, and ZY5 is about 28 days, and the appearance time of secondary protocorms of ZY4 is relatively late. Figure 2 and Figure 3 From the perspective of growth morphology, ZY1 is more inclined to expand horizontally into milky white protocorms, and the induction rate of milky white protocorms is higher, with the advantages of strong proliferation ability, fast germination speed and high accumulation of medicinal ingredients. The genetic traits of milky white protocorms are stable, which is extremely beneficial to rapid proliferation and germplasm resource preservation. Other varieties are more inclined to grow vertically into buds. Combined with the results of primary protocorm culture, taking into account the appearance time and induction rate of spherical corms, ZY1 (Jinkang No. 1) was selected as the most suitable variety for protocorm culture.

[0064] Table 2 Induction of secondary protocorms of Anoectochilus roxburghii from different provenances (n=100)

[0065] variety Time of appearance of bulbous tubers Induction rate of bulb-like stems ZY1 27.56±4.37 93±3.65 ZY2 29.44±4.62 90±2.89 ZY3 28.56±5.06 91±1.83 ZY4 34.44±5.62 74±5.3 ZY5 29.44±5.19 92±2.24

[0066] 3. Determine the culture conditions for culturing golden thread vine protocorms using bioreactors

[0067] 3.1 Test methods

[0068] The stably growing secondary protocorms are inoculated into a bioreactor and induced with the liquid culture medium provided by the present invention. The proliferation coefficient, fresh weight, dry weight and effective component content of the secondary protocorms are recorded, the effects of culture time and inoculation density on the secondary protocorms are investigated, and the conditions for culturing the secondary protocorms in the bioreactor are determined. The proliferation coefficient is calculated and the content of anoectochilin is detected.

[0069] 3.2 Test results

[0070] The proliferation coefficient and roxburghii glycoside content of Anoectochilus roxburghii bulbs with different culture time and inoculation amount are shown in Tables 3, 4, 5 and 6.

[0071] Table 3 Proliferation coefficient and roxburghii glycoside content of bulb-like stems cultured for 10 days at different inoculation doses (n=3)

[0072] Inoculation volume Proliferation coefficient Anoectochilus glycoside content 20g / L 0.48±0.08 6.20±0.16 40g / L 0.43±0.06 6.89±0.79 60g / L 0.43±0.05 7.98±0.14 80g / L 0.43±0.07 8.01±0.11

[0073] Table 4 Proliferation coefficient and roxburghii glycoside content of bulb-like stems cultured for 20 days at different inoculation doses (n=3)

[0074] Inoculation volume Proliferation coefficient Anoectochilus glycoside content 20g / L 0.77±0.12 7.34±0.28 40g / L 0.86±0.08 7.88±1.10 60g / L 0.93±0.07 7.8±0.27 80g / L 0.87±0.12 7.88+0.05

[0075] Table 5 Proliferation coefficient and roxburghii glycoside content of bulb-like stems cultured for 30 days at different inoculation doses (n=3)

[0076] Inoculation volume Proliferation coefficient Anoectochilus glycoside content 20g / L 1.10±0.08 7.35±0.51 40g / L 1.24±0.16 7.44±0.36 60g / L 1.32±0.14 8.51±0.56 80g / L 0.98±0.45 8.59±0.16

[0077] Table 6 Proliferation coefficient and roxburghii glycoside content of bulb-like stems cultured for 40 days at different inoculation doses (n=3)

[0078] Inoculation volume Proliferation coefficient Anoectochilus glycoside content 20g / L 1.15±0.07 6.96±0.28 40g / L 1.34±0.06 6.98±0.27 60g / L 1.40±0.08 6.65±0.29 80g / L 1.38±0.02 6.71±0.11

[0079] Under the conditions of ventilation volume 500mL / min and dark light culture, the proliferation coefficient of bulbous stems with different inoculation amounts and the content of roxburghii glycoside were studied. According to the data in Tables 3 to 6, the proliferation coefficient of all inoculation amounts increased significantly when the culture time increased from 10 days to 30 days. In particular, the proliferation coefficient of the inoculation amount of 60g / L reached 1.32 at 30 days, which was the highest among all conditions. However, when the culture time increased from 30 days to 40 days, the increase in the proliferation coefficient became slow and even stabilized. This shows that the proliferation rate was the fastest at 30 days, and after more than 30 days, the proliferation rate slowed down.

[0080] The content of anoectin increased significantly in all inoculum sizes when the culture time increased from 10 days to 30 days. In particular, the anoectin content of 60 g / L inoculum reached 8.51 ± 0.56 mg / g at 30 days. However, when the culture time increased from 30 days to 40 days, the anoectin content decreased significantly, indicating that the excessively long culture time led to a decrease in the content of anoectin.

[0081] When the inoculum size was lower than 60 g / L, the content of anoectin was low. For example, the content of anoectin was 7.44 ± 0.36 mg / g at 30 days with an inoculum size of 40 g / L, and 7.35 ± 0.51 mg / g at 30 days with an inoculum size of 20 g / L. This indicates that a higher inoculum size can effectively accumulate anoectin in a shorter culture time.

[0082] However, when the inoculum size is higher than 60g / L, the content of aureoside increases, but the growth rate is slow. For example, the aureoside content of 80g / L at 30 days is 8.59±0.16mg / g, which is only slightly higher than the inoculum size of 60g / L. Therefore, the economic benefit of the inoculum size of 80g / L is low. But at 40 days, the aureoside content of 80g / L inoculum is 6.71±0.11mg / g, which is significantly lower than the inoculum size of 60g / L. This shows that after the inoculum size exceeds 60g / L and the culture time is extended, the aureoside content not only does not increase significantly, but decreases.

[0083] Taking the proliferation coefficient and the content of anointing agent into consideration, the inoculation amount of 60 g / L performed best at 30 days, with a proliferation coefficient of up to 1.32, and the content of anointing agent significantly increased to 8.51±0.56 mg / g. This culture condition not only ensured the efficient proliferation of the bulbous stems, but also ensured the high content of anointing agent.

[0084] See also Figure 4-Figure 5 After proliferation, Jinkang No. 1 golden thread vine generated more lateral buds, which were more conducive to proliferation. Therefore, Jinkang No. 1 was selected as the proliferation variety of golden thread vine, and culturing it for 30 days with an inoculation amount of 60g / L was the best culture condition, which could effectively improve the proliferation efficiency and medicinal value of golden thread vine, and provide strong support for the large-scale production and medicinal development of golden thread vine.

[0085] The present invention is not limited to the above-mentioned optional implementation modes. Anyone can derive other various forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, all technical solutions that fall within the scope defined by the claims of the present invention fall within the protection scope of the present invention.

Claims

1. A method for culturing a roxburghii protocorm, characterized in that: include S1. Cultivation of primary protocorms of Anoectochilus roxburghii: Take a 1-1.5 cm stem segment of Jinkang No. 1 Anoectochilus roxburghii seedling, inoculate it into an induction medium, and culture it for 28-32 days at 23-27° C. and 16h / 8h light-dark cycle to obtain primary protocorms of Anoectochilus roxburghii; S2. Cultivation of secondary protocorms of Anoectochilus roxburghii: taking the buds of the primary protocorms of Anoectochilus roxburghii, cutting them into 2-4 mm slices, inoculating them into an induction medium, and culturing them for 58-62 days at 23-27° C. and 16h / 8h light / dark cycle to obtain secondary protocorms of Anoectochilus roxburghii; S3. Proliferation of secondary protocorms: inoculating the secondary protocorms of Anoectochilus roxburghii into a bioreactor at an inoculation amount of 55-65 g / L, inducing with an induction medium, and culturing for 28-32 days under aeration of 550-650 mL / min and dim light conditions to obtain protocorms of Anoectochilus roxburghii; The induction medium comprises MS medium, 2.5-3.5 mg / L 6-benzyladenine, 0.4-0.6 mg / L 1-naphthylacetic acid, 0.7-0.9 mg / L zeatin and 0.1-0.3 mg / L 2,4-dichlorophenoxyacetic acid.

2. The method for culturing a roxburghii-like protocorm according to claim 1, characterized in that: The induction medium includes MS medium, 3.2 mg / L 6-benzyladenine, 0.4 mg / L 1-naphthylacetic acid, 0.75 mg / L zeatin and 0.12 mg / L 2,4-dichlorophenoxyacetic acid.

3. The method for culturing a roxburghii-like protocorm according to claim 1, characterized in that: The induction medium includes MS medium, 2.5 mg / L 6-benzyladenine, 0.6 mg / L 1-naphthylacetic acid, 0.9 mg / L zeatin and 0.3 mg / L 2,4-dichlorophenoxyacetic acid.

4. The method for culturing a roxburghii-like protocorm according to claim 1, characterized in that: The induction medium includes MS medium, 3.0 mg / L 6-benzyladenine, 0.5 mg / L 1-naphthylacetic acid, 0.8 mg / L zeatin and 0.2 mg / L 2,4-dichlorophenoxyacetic acid.

5. The method for culturing a roxburghii-like protocorm according to claim 1, characterized in that: In step S1, the lighting condition of the photoperiod is 1500-2000 lux.

6. The method for culturing a roxburghii-like protocorm according to claim 5, characterized in that: In step S1, a 1.2 cm stem segment of a tissue culture seedling of Anoectochilus roxburghii was taken, inoculated into an induction medium, and cultured for 30 days at 25° C. and a 16 h / 8 h light / dark cycle to obtain a primary protocorm-like body of Anoectochilus roxburghii.

7. The method for culturing a roxburghii protocorm according to claim 1, characterized in that: In step S2, the buds of the primary protocorm-like bodies of A. roxburghii are taken, cut into 3 mm thin slices, inoculated into an induction culture medium, and cultured for 60 days at 25° C. and a 16h / 8h light / dark cycle to obtain secondary protocorms-like bodies of A. roxburghii.

8. The method for culturing a roxburghii protocorm according to claim 1, characterized in that: In step S3, the secondary protocorms of Anoectochilus roxburghii are inoculated into a bioreactor at an inoculation amount of 55 g / L, induced with an induction medium, and cultured for 32 days under aeration of 620 mL / min and dark light conditions to obtain protocorms of Anoectochilus roxburghii.

9. The method for culturing a roxburghii-like protocorm according to claim 1, characterized in that: In step S3, the secondary protocorms of A. roxburghii are inoculated into a bioreactor at an inoculation amount of 60 g / L, induced with an induction medium, and cultured for 28 days under aeration of 600 mL / min and dark light conditions to obtain protocorms of A. roxburghii.

10. The method for culturing a roxburghii protocorm according to claim 1, characterized in that: In step S3, the secondary protocorms of A. roxburghii are inoculated into a bioreactor at an inoculation amount of 60 g / L, induced with an induction medium, and cultured for 30 days under aeration of 600 mL / min and dark light conditions to obtain protocorms of A. roxburghii.

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