Culture medium and culture method of orchid stem cells

Abstract

The invention discloses a culture medium and a culture method of orchid stem cells. The culture medium comprises a basic culture medium, and 100-300 mg / L of lanthanum nitrate is added into the basic culture medium; the basic culture medium is selected from one of an MS culture medium and a B5 culture medium. The basic culture medium is an MS culture medium, 0.9-1.2 mg / L of amphotericin B and a double antibody with the volume concentration of 0.8%-1.3% are further added, and the double antibody is a 100X mycillin mixed solution, 0.9-1.1 mg / L of 6-BA and 0.9-1.2 mg / L of NAA; or the basic culture medium is a B5 culture medium, and 0.099 mg / L to 0.130 mg / L of 6-BA, 12 mg / L to 17 mg / L of cane sugar, 0.49 mg / L to 0.54 mg / L of methyl jasmonate and 0.098 mg / L to 0.120 mg / L of triacontanol are also added into the basic culture medium. Lanthanum nitrate with the concentration of 100-300 mg / L is added into the culture medium for culturing the orchid stem cells, and experiments show that a large number of stem cells can be cultured within a short time by adding lanthanum nitrate with the concentration range, the cell dispersion degree is increased, the number of the cells is increased, and a foundation is provided for subculture multiplication culture in the next step.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant tissue culture, and specifically relates to a culture medium and a culture method for orchid plant stem cells. Background Art

[0002] Orchidaceae plants are one of the largest families of angiosperms, with approximately 700 genera and 20,000 - 35,000 species worldwide, widely distributed in various terrestrial ecosystems except the polar regions and extremely arid desert areas, with the most in tropical regions. In terms of plant system evolution, as a relatively specialized group, Orchidaceae plants are one of the hot groups in biological research. China is one of the countries richest in wild orchidaceae plants, with a series of evolutionary groups from primitive types to advanced types and complex and diverse geographical distribution types.

[0003] Orchidaceae plants include ornamental orchids, Dendrobium officinale, Dendrobium loddigesii, Dendrobium fimbriatum, Dendrobium chrysanthum, Dendrobium nobile, etc.

[0004] Dendrobium huoshanense, commonly known as "Mihu", is a perennial herbaceous plant of the genus Dendrobium in Orchidaceae and is a typical representative of Dendrobium officinale. Its flower stem is thicker, cylindrical; its leaves are thinner, oblong, with fluff under the leaf tips; its flowers are larger, polygon-shaped, red, with a slightly grassy aroma; its roots are thinner, tender white, and smooth on the surface. The flowering period is from May to June, and the fruiting period is from July to August. According to "Baicao Jing": There is a type of Dendrobium recently, as thin as a lampwick, with a sweet taste when chewed and slightly slippery saliva. It comes from Huoshan County, Lu'an City and Yingzhou Prefecture, and is called "Dendrobium huoshanense". The growth of Dendrobium huoshanense has extremely strict environmental requirements. Coupled with the collection by medicinal farmers in past dynasties and the changes in the modern ecological environment, the wild resources of Dendrobium huoshanense are on the verge of extinction, and now only a very small amount has been transplanted to the Changchong Medicinal Materials Farm in Huoshan County. Although Dendrobium huoshanense has many flowers, it has few fruits, and the seeds of Dendrobium huoshanense have undifferentiated embryos and no endosperm, resulting in a very low germination rate, unable to meet the needs of large-scale artificial cultivation. Dendrobium huoshanense is native to Huoshan County, Anhui Province in the Dabie Mountains, and is mainly distributed in Taiwan, Guangdong, Guangxi, Hubei, Jiangsu and Zhejiang. It likes shade and is afraid of direct sunlight, growing densely in groups in warm, rainy and foggy valleys, and mostly grows on the cliffs of sheltered and shady mountain gullies.

[0005] The main propagation methods of Orchidaceae plants are sowing propagation and cutting propagation. In actual life, sowing propagation is often used, and it can also be sown with the help of external forces. There is also seed tissue culture, that is, fruits harvested from healthy plants are used for tissue culture and division propagation, and division propagation is carried out from mother plants over 3 years old.

[0006] Due to being affected by various factors such as the slow growth of medicinal plants, limited natural sources, the raw materials being easily polluted by the environment and toxins, and the reduction of active ingredients, it is difficult to meet the needs of industrial production, further exacerbating the survival threat of medicinal plants.

[0007] At present, the cell engineering research on Dendrobium huoshanense is still limited to callus and extraction of active ingredients. For example, the Chinese patent with the application number CN201110179435.3 discloses a method for somatic embryo development of Dendrobium huoshanense. The method includes the following operation steps: (1) induction of axenic test-tube seedlings; (2) induction of embryogenic callus; (3) induction of somatic embryos; (4) synchronous proliferation of somatic embryos to obtain a large number of somatic embryos of Dendrobium huoshanense. By taking the morphological lower end of the stem segment with nodes of axenic test-tube seedlings as the explant and controlling the redox potential of the induction medium, embryogenic callus of Dendrobium huoshanense can be induced without adding exogenous hormones, avoiding the harm to human health caused by the enrichment of exogenous hormones into intact plants through somatic embryos. By controlling the environmental humidity, embryogenic callus develops into somatic embryos, with the consistency of spherical somatic embryos reaching over 90% and the survival rate of regenerated plants reaching over 85%. The technology disclosed is a method for somatic embryo development. Somatic embryos are embryos developed from somatic cells and do not involve the culture method of stem cells.

[0008] The Chinese patent with the application number CN201610353012.1 discloses a method for producing Dendrobium alkaloids by suspension culture of cells, which is a method for producing Dendrobium alkaloids by suspension culture of Dendrobium huoshanense cells in a cell culture reactor, including the following steps: (1) single cell preparation; (2) single cell line culture; (3) cell mass scale-up culture; (4) extraction of Dendrobium alkaloids: When the cell density reaches 60,000 - 80,000 cells / ml, collect Dendrobium huoshanense cells, lower the culture temperature to 7 - 10 °C, continue culturing for 5 - 7 days, and extract Dendrobium alkaloids. It can greatly shorten the cell culture cycle, improve the yield of Dendrobium alkaloid products, and has a simple and stable process, suitable for industrial production. This technology also does not involve the culture method of stem cells.

[0009] The Chinese patent with the application number CN201510770456.0 discloses a method for culturing Dendrobium officinale stem cells. Inoculate small pieces of Dendrobium officinale root tips onto a screening medium and culture them in the dark for 5 - 10 days to obtain Dendrobium officinale stem cells derived from the cambium, and then transfer them to a liquid medium for culturing for 3 days. The liquid medium is B5 medium containing 0.1 mg / L of 6 - BA, 15 mg / L of sucrose, 0.5 mg / L of methyl jasmonate, and 0.1 mg / L of triacontanol. The stem cells cultured in this scheme are cambium stem cells and it takes 8 - 13 days, which is a long time.

[0010] Plant stem cells are undifferentiated cells with eternal vitality in plants, mainly located in the shoot apical meristem, root apical meristem and vascular cambium of the plant body. Plant stem cells are the root of plant vitality, enabling plants to grow continuously over hundreds of years. Plant stem cells are the most primitive totipotent stem cells in plants and have great advantages compared with the cambium (CMC) stem cells of plants. Plant stem cells are undifferentiated, with strong division ability, extremely high cell proliferation rate, extremely high cell activity, high and stable content of secondary metabolites; while the cambium stem cells of plants are also derived from plant stem cells and have achieved initial differentiation. Relatively speaking, their division ability is weak, the cell proliferation rate and cell activity are low, and the secondary metabolite content will decrease generation by generation. Therefore, it is necessary to develop plant stem cell technology to adapt to large-scale production.

[0011] Thus, there is currently no research on the stem cells of Orchidaceae plants such as Dendrobium huoshanense. Therefore, it is urgent to develop a method for stably obtaining Orchidaceae plant stem cells with a variety of active ingredients.

[0012] In view of this, the present invention is specifically proposed. Summary of the Invention

[0013] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a culture medium and a culture method for Orchidaceae plant stem cells. Lanthanum nitrate, a rare earth element, is added to the culture medium of the present invention, which can culture a large number of stem cells in a short time.

[0014] To solve the above technical problems, the basic concept of the technical solution adopted by the present invention is:

[0015] The present invention provides a culture medium for Orchidaceae plant stem cells, the culture medium includes a basal medium, and 100-300 mg / L of lanthanum nitrate is added to the basal medium; the basal medium is selected from one of MS medium and B5 medium.

[0016] Rare earth elements have special properties of promoting crop yield increase, enhancing plant stress resistance, and improving crop quality. The area of crops applying rare earth element compound fertilizers is getting wider and wider. At the same time, due to the continuous entry of rare earth products into the environment and food chains, the impacts on the ecological environment, the growth of animals and plants, human health and their long-term effects have attracted extensive attention from the scientific community at home and abroad. And rare earth elements are one of the effective regulators of plant growth and metabolite synthesis. Appropriate concentrations of rare earth elements can significantly promote plant cell growth and metabolite synthesis.

[0017] In the culture medium for culturing orchid plant stem cells of the present invention, lanthanum nitrate with a concentration of 100 - 300 mg / L is added. Through experiments, it is found that adding lanthanum nitrate within this concentration range can culture a large number of orchid plant stem cells in a relatively short time, and improve the degree of cell dispersion and the number of cells, providing a basis for the next subculture and proliferation culture.

[0018] The present invention has conducted experiments on the culture of stem cells of various Araliaceae plants, such as Dendrobium huoshanense (Mi Hu), Dendrobium loddigesii, Dendrobium fimbriatum, Dendrobium chrysanthum, Dendrobium nobile, Dendrobium officinale, etc. It is found that the culture medium containing lanthanum nitrate of the present invention has universality for the culture of orchid plant stem cells, and can all culture a large number of stem cells in a relatively short time.

[0019] In a further embodiment, the basal medium is MS medium, and it also contains amphotericin B at a concentration of 0.9 - 1.2 mg / L, double antibody with a volume concentration of 0.8% - 1.3% (the double antibody is 100X penicillin - streptomycin mixture), 6 - BA at a concentration of 0.9 - 1.1 mg / L, and NAA at a concentration of 0.9 - 1.2 mg / L.

[0020] In a further embodiment, the MS medium contains 1.0 mg / L of amphotericin B, double antibody with a volume concentration of 1.0%, 1.0 mg / L of 6 - BA, and 1.0 mg / L of NAA.

[0021] The stock solution of the double antibody is a penicillin - streptomycin mixed solution (100×), and the double antibody with a volume concentration of 1% is diluted 100 times from the stock solution.

[0022] The amphotericin B added to the culture medium can play an antifungal role, and the double antibody can play an antibacterial role to prevent the culture medium from being contaminated with bacteria; 6 - BA (6 - benzylaminopurine) and NAA (naphthaleneacetic acid) can promote differentiation. Therefore, the various components in the culture medium act synergistically, can well promote the culture of stem cells, and increase the culture amount of stem cells.

[0023] In a further embodiment, the basal medium is B5 medium, and it also contains 0.099 - 0.130 mg / L of 6 - BA, 12 - 17 mg / L of sucrose, 0.49 - 0.54 mg / L of methyl jasmonate, and 0.098 - 0.120 mg / L of triacontanol.

[0024] In a further embodiment, the B5 medium contains 0.100 mg / L of 6 - BA, 15 mg / L of sucrose, 0.50 mg / L of methyl jasmonate, and 0.100 mg / L of triacontanol.

[0025] 6-BA (6-benzylaminopurine), methyl jasmonate and triacontanol can further promote differentiation. Therefore, the components in the culture medium act synergistically to promote the culture of stem cells and increase the culture yield of stem cells.

[0026] The present invention also provides a method for culturing orchid stem cells, comprising:

[0027] (1) Pretreatment of explants: Take the tender stems of orchid plants, wash, disinfect, and set aside;

[0028] (2) Obtaining of stem cells: Dissect the meristem from the stem tip after being treated in step (1), transfer it to a culture medium for culture. The culture medium comprises a basal medium, and 100 - 300 mg / L of lanthanum nitrate is added to the basal medium; the basal medium is selected from one of MS medium and B5 medium; after the culture is completed, filter to obtain a filtrate containing stem cells;

[0029] (3) Subculture: Transfer the filtrate containing stem cells obtained in step (2) to a culture medium for culture. After the culture is completed, divide the culture medium containing stem cells into several portions, transfer them to several culture containers, and then add the culture medium respectively for subculture;

[0030] (4) Collection: Centrifuge the stem cell culture solution obtained in step (3), discard the supernatant, and obtain the orchid stem cells.

[0031] In a further embodiment, in steps (2) and (3),

[0032] the culture medium comprises: the basal medium is MS medium, wherein 100 - 300 mg / L of lanthanum nitrate, 0.9 - 1.2 mg / L of amphotericin B, a double antibody with a volume concentration of 0.8% - 1.3% (the double antibody is a 100X penicillin-streptomycin mixture), 0.9 - 1.1 mg / L of 6-BA and 0.9 - 1.2 mg / L of NAA are added;

[0033] Or, the culture medium comprises: the basal medium is B5 medium, wherein 100 - 300 mg / L of lanthanum nitrate, 0.099 - 0.130 mg / L of 6-BA, 12 - 17 mg / L of sucrose, 0.49 - 0.54 mg / L of methyl jasmonate and 0.098 - 0.120 mg / L of triacontanol are added.

[0034] In a further embodiment, control the pH value of the culture medium to be 5.8 - 6.8; preferably 6.2.

[0035] Preferably, the culture medium in step (3) is the same as the culture medium in step (2);

[0036] Preferably, in step (3), subculture is carried out once or multiple times.

[0037] In a further embodiment, in step (1), the cleaning and disinfection include:

[0038] The shoot tip is rinsed with clear water, then washed with an aqueous alcohol solution with a volume concentration of 75%, rinsed with sterile water, then disinfected with a disinfectant containing sodium hypochlorite, and then rinsed with sterile water again.

[0039] In a further embodiment, in steps (2) and (3), the temperature for culturing is 20 - 25 °C, the shaker speed is 80 - 200 r / min, and the culturing time is 3 - 7 days.

[0040] As a specific embodiment, in step (2), the temperature for culturing is 22 °C, the shaker speed is 110 r / min, and the culturing time is 3 days.

[0041] As a specific embodiment, in step (3), the temperature for culturing is 22 °C, the shaker speed is 110 r / min, and the culturing time for subculture once is 2 days.

[0042] In a further embodiment, the Orchidaceae plants described in the present invention include ornamental orchids, Dendrobium officinale, Dendrobium loddigesii, Dendrobium fimbriatum, Dendrobium chrysanthum, Dendrobium nobile, Dendrobium huoshanense, Dendrobium moniliforme, Dendrobium strongylanthum, Dendrobium wardianum, Dendrobium aduncum, Dendrobium denneanum.

[0043] As a specific embodiment, the method for culturing Orchidaceae plant stem cells specifically includes:

[0044] (1) Pretreatment of young stems: First, take the young stems of Orchidaceae plants, rinse them with clear water for 3 h, carefully brush the parts that are not easy to clean with a brush to remove dust and other attachments, then wash and disinfect them with an aqueous alcohol solution with a volume concentration of 75% for 20 s, then rinse with sterile water 3 times, next disinfect them with a disinfectant containing sodium hypochlorite for 5 min, and finally rinse with sterile water 3 times for standby;

[0045] (2) Obtaining of stem cells: After the young stems disinfected in step (1) are cut off the shoot tip with a surgical blade in a biosafety cabinet, the shoot tip is peeled layer by layer with a dissecting needle and forceps, leaving 5 - 6 leaf primordia, then cut off at 0.5 mm from the shoot tip with a surgical blade, and in a sufficient interval space, the shoot tip with the meristem of Dendrobium huoshanense can be picked out with a dissecting needle, and then the separated shoot tip meristem is put into a triangular flask containing a culture medium, and the triangular flask is placed in a vertical constant temperature oscillator with a speed of 110 r / min and a temperature of 22 °C for shaking culture, and the culturing time is 3 days; after the culturing is completed, first filter out larger flakes and fragments with a No. 2 sieve, and then perform secondary filtration separation with a 40 - μm cell filter membrane, and the filtrate is the filtrate containing stem cells;

[0046] (3) Expansion culture of stem cells: Transfer the finally filtered filtrate containing stem cells into an Erlenmeyer flask, add the culture medium, place the Erlenmeyer flask on a vertical constant temperature shaker with a rotation speed of 110 r / min and a temperature of 22 °C, and perform shaking culture for 2 days. After the culture is completed, transfer the culture medium containing stem cells evenly from 1 Erlenmeyer flask to 3 Erlenmeyer flasks, continue to add the culture medium to each Erlenmeyer flask until the amount of the culture medium reaches about 1 / 3 of the position, and perform subculture on a vertical constant temperature shaker with a rotation speed of 110 r / min and a temperature of 22 °C;

[0047] (4) Collection of stem cells: Perform subculture on stem cells and collect them. Pour the culture medium containing stem cells into a centrifuge bottle, balance it, place it in a centrifuge with a rotation speed of 2200 r / min and a centrifugation time of 10 minutes. After centrifugation, discard the supernatant, and combine the precipitates obtained by centrifuging the culture medium in each Erlenmeyer flask to obtain the stem cells of Orchidaceae plants.

[0048] After adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art.

[0049] 1. In the culture medium for culturing stem cells of Orchidaceae plants of the present invention, lanthanum nitrate with a concentration of 100 - 300 mg / L is added. It is found through experiments that adding lanthanum nitrate within this concentration range can culture a large number of stem cells in a relatively short time, and improve the cell dispersion degree and quantity, providing a basis for the next subculture and proliferation culture.

[0050] 2. The present invention has conducted experiments on the culture of stem cells of various Araliaceae plants, such as Dendrobium huoshanense (Mi Hu), Dendrobium loddigesii, Dendrobium fimbriatum, Dendrobium chrysanthum, Dendrobium nobile, Dendrobium officinale, etc. It is found that the culture medium containing lanthanum nitrate of the present invention has universality for the culture of stem cells of Orchidaceae plants, and can culture a large number of stem cells in a relatively short time. Specific embodiments

[0051] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below in conjunction with the embodiments of the present invention. The following embodiments are used to illustrate the present invention but are not used to limit the scope of the present invention.

[0052] Embodiment 1

[0053] This embodiment relates to the culture and collection method of Dendrobium huoshanense stem cells under the condition of B5 culture medium added with lanthanum nitrate. The specific steps are as follows:

[0054] (1) Pretreatment of young stems: First, take the young stems of Dendrobium officinale Kimura et Migo, rinse them with clean water for 3 h, carefully brush the parts that are not easy to clean with a brush to remove dust and other attachments, then clean and disinfect them with an aqueous alcohol solution with a volume concentration of 75% for 20 s, then rinse them 3 times with sterile water, next disinfect them with a disinfectant containing sodium hypochlorite for 5 min, and finally rinse them 3 times with sterile water for standby;

[0055] (2) Obtaining of stem cells: Cut off the stem tip of the disinfected young stems in step (1) with a surgical blade in a biosafety cabinet, layer by layer peel the stem tip with a dissecting needle and forceps, leave 5 - 6 leaf primordia, then cut it at 0.5 mm from the stem tip with a surgical blade, and in a sufficient interval space, the stem tip with the meristem of Dendrobium officinale can be picked out with a dissecting needle, and then transfer the separated stem tip meristem into a triangular flask containing a culture medium for culture. The culture medium is B5 medium containing 0.100 mg / L of 6 - BA, 15 mg / L of sucrose, 0.50 mg / L of methyl jasmonate and 0.100 mg / L of triacontanol. In addition, the culture medium is added with lanthanum nitrate with a final concentration of 300 mg / L, and the pH value of the culture medium is 6.2; Place the triangular flask on a vertical constant temperature oscillator, with a rotation speed of 110 r / min and a temperature of 22 °C, and perform shaking culture for 3 days; After the culture is completed, first filter out the larger flakes and fragments with a No. 2 sieve, and then perform secondary filtration separation with a 40 - um cell filter membrane. The filtrate is the filtrate containing stem cells;

[0056] (3) Sub - culture of stem cells: Transfer the finally filtered filtrate containing stem cells into a triangular flask, and add B5 medium containing 0.100 mg / L of 6 - BA, 15 mg / L of sucrose, 0.50 mg / L of methyl jasmonate and 0.100 mg / L of triacontanol. In addition, the culture medium is respectively added with lanthanum nitrate with a final concentration of 300 mg / L, and place it on a vertical constant temperature oscillator, with a rotation speed of 110 r / min and a temperature of 22 °C, and perform shaking culture for 2 days. After the culture is completed, evenly transfer the culture medium containing stem cells from 1 triangular flask to 3 triangular flasks, and continue to add B5 medium containing 0.100 mg / L of 6 - BA, 15 mg / L of sucrose, 0.50 mg / L of methyl jasmonate, 0.100 mg / L of triacontanol and lanthanum nitrate with a final concentration of 300 mg / L to each triangular flask until the amount of the culture medium in each triangular flask reaches about 1 / 3 of the position, and perform sub - culture on a vertical constant temperature oscillator, with a rotation speed of 110 r / min and a temperature of 22 °C;

[0057] (4) Collection of stem cells: The stem cells are subcultured for 48 hours and then collected. The culture medium containing the stem cells is poured into a centrifuge bottle. After balancing, it is placed in a centrifuge at a rotational speed of 2200 r / min for 10 minutes. After centrifugation, the supernatant is discarded to obtain Dendrobium officinale stem cells. Alternatively, the obtained stem cells can be freeze-dried or microwave-dried. After complete drying, they are pulverized with a small pulverizer and sieved to obtain Dendrobium officinale stem cell powder.

[0058] Example 2

[0059] This example relates to a method for culturing and collecting Dendrobium officinale stem cells under the condition of MS medium added with lanthanum nitrate. The specific steps are as follows:

[0060] (1) Pretreatment of tender stems: First, take the tender stems of Dendrobium officinale and rinse them with clean water for 3 hours. Carefully brush the parts that are not easy to clean with a brush to remove dust and other attachments. Then, clean and disinfect them with an aqueous alcohol solution with a volume concentration of 75% for 20 seconds, and then rinse them 3 times with sterile water. Next, disinfect them with a disinfectant containing sodium hypochlorite for 5 minutes, and finally rinse them 3 times with sterile water for standby.

[0061] (2) Obtaining of stem cells: Cut off the shoot tips of the disinfected tender stems in step (1) with a surgical blade in a biosafety cabinet. Layer by layer peel the shoot tips with a dissecting needle and forceps, leaving 5 - 6 leaf primordia. Then cut at 0.5 mm from the shoot tip with a surgical blade. In a sufficient interval space, the shoot tips with Dendrobium officinale meristem can be picked out with a dissecting needle. Then place the separated shoot tip meristems into 3 Erlenmeyer flasks containing the culture medium for culture. The culture medium is MS medium containing 1.0 mg / L amphotericin B, 1.0% (v / v) double antibody (100X penicillin-streptomycin mixture), 1.0 mg / L 6-BA, and 1.0 mg / L NAA. Lanthanum nitrate with final concentration gradients of 100 mg / L, 200 mg / L, and 300 mg / L is respectively added to the culture medium. Place the Erlenmeyer flasks in a vertical constant temperature oscillator at a rotational speed of 110 r / min and a temperature of 22 °C for shaking culture for 3 days. After the culture is completed, first filter out larger flakes and fragments with a No. 2 sieve, and then perform secondary filtration separation with a 40 μm cell filter membrane. The filtrate is the filtrate containing stem cells.

[0062] (3) Expansion culture of stem cells: Transfer the finally filtered filtrate containing stem cells into an Erlenmeyer flask, and add MS medium containing amphotericin B at a concentration of 1.0 mg / L, double antibody at a volume concentration of 1.0%, 6-BA at 1.0 mg / L, and NAA at 1.0 mg / L. Lanthanum nitrate with final concentration gradients of 100 mg / L, 200 mg / L, and 300 mg / L is also added to the medium. Place the Erlenmeyer flask on a vertical constant temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C for shaking culture for 2 days. After the culture, evenly transfer the medium containing stem cells from 1 Erlenmeyer flask to 3 Erlenmeyer flasks, and continue to add MS medium containing amphotericin B at a concentration of 1.0 mg / L, double antibody at a volume concentration of 1.0%, 6-BA at 1.0 mg / L, and NAA at 1.0 mg / L (lanthanum nitrate with final concentration gradients of 100 mg / L, 200 mg / L, and 300 mg / L is added to the medium) until the amount of medium in each Erlenmeyer flask reaches about 1 / 3 position. Then, place it on a vertical constant temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C for subculture;

[0063] (4) Collection of stem cells: After 48 h of subculture of stem cells, collect them. Pour the medium containing stem cells into a centrifuge bottle, balance it, and then place it in a centrifuge with a rotation speed of 2200 r / min and a centrifugation time of 10 minutes. After centrifugation, discard the supernatant to obtain the stem cells of Dendrobium officinale. The obtained stem cells can also be freeze-dried or microwave-dried. After complete drying, use a small crusher to crush and sieve them to obtain the stem cell powder of Dendrobium officinale. Collect the stem cells of Dendrobium officinale cultured in media with different concentrations of lanthanum nitrate respectively.

[0064] Experimental Example 1

[0065] This experimental example involves the identification of the stem cells of Dendrobium officinale obtained in Examples 1 and 2 and the experiment on the effect of lanthanum nitrate on the stem cells of Dendrobium officinale.

[0066] (1) Experiment on the effect of lanthanum nitrate on the stem cells of Dendrobium officinale:

[0067] Set Control Example 1: Different from Example 1, lanthanum nitrate is not added to the used B5 medium, and the others are the same as Example 1.

[0068] Control Example 2: Different from Example 2, lanthanum nitrate is not added to the used MS medium, and the others are the same as Example 2.

[0069] Weigh the finally obtained stem cells of Dendrobium officinale in the above Examples 1 and 2 and Control Examples 1 and 2 respectively. Conduct 5 experiments for each group and finally take the average value. The results are shown in Table 1.

[0070] Table 1 Comparison results of the weights of the stem cells of Dendrobium officinale

[0071] Example 1 Comparative Example 1 Example 2 Example 2 Example 2 Comparative Example 2 Lanthanum nitrate concentration / mg / L 300 0 100 200 300 0 Culture medium type B5 B5 MS MS MS MS Weight / g 94.56 78.22 108.56 114.44 122.13 82.43

[0072] As can be seen from Table 1, the weight ratios of the Dendrobium officinale stem cells obtained in Examples 1 and 2 are larger than those in Comparative Examples 1 and 2, indicating that the culture medium supplemented with lanthanum nitrate at a concentration of 100-300 mg / L can cultivate a large number of stem cells in a relatively short time. The stem cells obtained from the culture media with different concentrations of lanthanum nitrate in Example 2 are superior to those in Example 1, indicating that the composition and ratio of the culture medium in Example 2 are more suitable for cultivating a large number of Dendrobium officinale stem cells in a short time.

[0073] (2) Identification of stem cells

[0074] Stem cells can be identified by staining microscopy. That is, the stem cells obtained in the above examples and comparative examples are centrifuged, the supernatant is removed, phosphate buffer solution with pH 7.2 is added, and then 10 μL of stem cells are taken and smeared on a clean glass slide. Then, 3-4 drops of 0.1% neutral red staining solution are added to the stem cells on the glass slide to completely cover the stem cells, and staining is carried out at room temperature for 30 min; after the staining is completed, gently rinse along the edge of the staining solution with running water, after rinsing clean, blot the moisture on the edge with filter paper, cover the cover glass, and immediately observe under a microscope.

[0075] It can be clearly observed through microscopy that the growth condition of the Dendrobium officinale stem cells obtained in Example 2 is better than that of the stem cells obtained in other examples and comparative examples, the dispersibility is stronger, and the concentration is proportional to the growth condition.

[0076] Example 3

[0077] The orchid plant used in this example is Dendrobium officinale, and it relates to a method for culturing Dendrobium officinale stem cells. The specific steps are as follows:

[0078] (1) Pretreatment of tender stems: First, take the tender stems of Dendrobium officinale and rinse them with clean water for 3 h, and carefully brush the parts that are not easy to clean with a brush to remove dust and other attachments. Then, clean and disinfect them with an aqueous alcohol solution with a volume concentration of 75% for 20 s, and then rinse them with sterile water 3 times. Next, disinfect them with a disinfectant containing sodium hypochlorite for 5 min, and finally rinse them with sterile water 3 times for standby;

[0079] (2) Obtaining of stem cells: After disinfection in step (1), cut off the shoot tip of the tender stem with a surgical blade in a biosafety cabinet. Layer by layer peel the shoot tip with a dissecting needle and forceps, leaving 5 - 6 leaf primordia. Then cut it at 0.5 mm from the shoot tip with a surgical blade. In a sufficient interval space, pick out the shoot tip with the Dendrobium officinale Kimura et Migo meristem with a dissecting needle. Then transfer the isolated shoot tip meristem into a triangular flask containing a culture medium for culturing. The culture medium is B5 medium containing 0.099 mg / L of 6 - BA, 12 mg / L of sucrose, 0.49 mg / L of methyl jasmonate and 0.098 mg / L of triacontanol. Additionally, lanthanum nitrate with a concentration of 300 mg / L is added to the culture medium. The pH value of the culture medium is 6.2. Place the triangular flask on a vertical constant temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C for shaking culture for 3 days. After the culture is completed, first filter out larger flakes and debris with a No. 2 sieve, and then perform secondary filtration separation with a 40 - μm cell filter membrane. The filtrate is the filtrate containing stem cells;

[0080] (3) Sub - culture expansion of stem cells: Transfer the finally filtered filtrate containing stem cells into a triangular flask, and add B5 medium containing 0.099 mg / L of 6 - BA, 12 mg / L of sucrose, 0.49 mg / L of methyl jasmonate and 0.098 mg / L of triacontanol. Additionally, lanthanum nitrate with a concentration of 300 mg / L is added to the culture medium. Place the triangular flask on a vertical constant temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C for shaking culture for 2 days. After the culture is completed, evenly transfer the culture medium containing stem cells from 1 triangular flask to 3 triangular flasks, and continue to add to them B5 medium that has already been added with 0.099 mg / L of 6 - BA, 12 mg / L of sucrose, 0.49 mg / L of methyl jasmonate, 0.098 mg / L of triacontanol and lanthanum nitrate with a concentration of 300 mg / L until the amount of the culture medium in each triangular flask reaches about 1 / 3 of the position. Place it on a vertical constant temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C for sub - culture;

[0081] (4) Collection of stem cells: After sub - culturing the stem cells for 48 h, collect them. Pour the culture medium containing stem cells into a centrifuge bottle, balance it, and then place it in a centrifuge with a rotation speed of 2200 r / min and a centrifugation time of 10 minutes. After centrifugation, discard the supernatant to obtain the Dendrobium officinale Kimura et Migo stem cells. Freeze - dry or microwave - dry the obtained stem cells. After complete drying, use a small crusher to crush and sieve them to obtain Dendrobium officinale Kimura et Migo stem cell powder.

[0082] Example 4

[0083] The orchid plant used in this embodiment is Dendrobium officinale var. moniliforme, and it involves a method for culturing Dendrobium officinale var. moniliforme stem cells. The specific steps are as follows:

[0084] (1) Pretreatment of tender stems: First, take the tender stems of Dendrobium officinale var. moniliforme and rinse them with clean water for 3 h. Carefully brush the parts that are not easy to clean with a brush to remove dust and other attachments. Then, clean and disinfect them with an aqueous alcohol solution with a volume concentration of 75% for 20 s, and then rinse them 3 times with sterile water. Next, disinfect them with a disinfectant containing sodium hypochlorite for 5 min, and finally rinse them 3 times with sterile water for standby;

[0085] (2) Obtaining stem cells: Cut off the shoot tips of the disinfected tender stems in step (1) with a surgical blade in a biosafety cabinet. Layer by layer peel the shoot tips with a dissecting needle and forceps, leaving 5 - 6 leaf primordia. Then, cut at 0.5 mm from the shoot tip with a surgical blade. In a sufficient interval space, the shoot tips with Dendrobium officinale var. moniliforme meristem can be picked out with a dissecting needle. Then, transfer the isolated shoot tip meristem into a triangular flask containing a culture medium for cultivation. The culture medium is B5 medium containing 0.130 mg / L of 6 - BA, 17 mg / L of sucrose, 0.54 mg / L of methyl jasmonate, and 0.120 mg / L of triacontanol. In addition, lanthanum nitrate with a concentration of 300 mg / L is added to the culture medium, and the pH value of the culture medium is 6.2. Place the triangular flask on a vertical constant temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C for shaking culture. The culture time is 3 days. After the culture is completed, first filter out the larger flakes and fragments with a No. 2 sieve, and then perform secondary filtration separation with a 40 - um cell filter membrane. The filtrate is the filtrate containing stem cells;

[0086] (3) Sub - culture of stem cells: Transfer the finally filtered filtrate containing stem cells into a triangular flask, and add B5 medium containing 0.130 mg / L of 6 - BA, 17 mg / L of sucrose, 0.54 mg / L of methyl jasmonate, and 0.120 mg / L of triacontanol. In addition, lanthanum nitrate with a concentration of 300 mg / L is added to the culture medium. Place the triangular flask on a vertical constant temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C for shaking culture. The culture time is 2 days. After the culture is completed, evenly transfer the culture medium containing stem cells from 1 triangular flask to 3 triangular flasks, and continue to add B5 medium that has already been added with 0.130 mg / L of 6 - BA, 17 mg / L of sucrose, 0.54 mg / L of methyl jasmonate, 0.120 mg / L of triacontanol, and lanthanum nitrate with a concentration of 300 mg / L until the amount of the culture medium in each triangular flask reaches about 1 / 3 of the position. Perform sub - culture on a vertical constant temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C;

[0087] (4) Collection of stem cells: The stem cells are subcultured for 48 hours and then collected. The culture medium containing the stem cells is poured into a centrifuge bottle. After balancing, it is placed in a centrifuge at a rotational speed of 2200 r / min for 10 minutes. After centrifugation, the supernatant is discarded to obtain the stem cells of Dendrobium officinale. The obtained stem cells are freeze-dried or microwave-dried. After complete drying, they are pulverized with a small pulverizer and sieved to obtain the Dendrobium officinale stem cell powder.

[0088] Example 5

[0089] The orchid plant used in this example is Dendrobium officinale, and it involves a method for culturing Dendrobium officinale stem cells. The specific steps are as follows:

[0090] (1) Pretreatment of tender stems: First, take the tender stems of Dendrobium officinale and rinse them with clean water for 3 hours. Carefully brush the parts that are not easy to clean with a brush to remove dust and other attachments. Then, clean and disinfect them with an aqueous alcohol solution with a volume concentration of 75% for 20 seconds, and then rinse them 3 times with sterile water. Next, disinfect them with a disinfectant containing sodium hypochlorite for 5 minutes, and finally rinse them 3 times with sterile water for standby;

[0091] (2) Obtaining stem cells: Cut off the stem tip of the disinfected tender stems in step (1) with a surgical blade in a biosafety cabinet. Layer by layer peel the stem tip with a dissecting needle and forceps, leaving 5 - 6 leaf primordia. Then cut it at 0.5 mm from the stem tip with a surgical blade. In a sufficient interval space, the stem tip with the Dendrobium officinale meristem can be picked out with a dissecting needle. Then, place the separated stem tip meristem into 3 Erlenmeyer flasks containing the culture medium for culture. The culture medium is an MS medium containing amphotericin B at a concentration of 0.9 mg / L, double antibiotics (100X penicillin-streptomycin mixture) at a mass concentration of 0.8%, 6-BA at 0.9 mg / L, and NAA at 0.9 mg / L. In addition, lanthanum nitrate at a concentration of 300 mg / L is added to the culture medium respectively. The pH value of the culture medium is 6.2. Place the Erlenmeyer flasks on a vertical constant temperature oscillator with a rotational speed of 110 r / min and a temperature of 22 °C, and perform shaking culture for 3 days respectively. After the culture is completed, first filter out the larger flakes and fragments with a No. 2 sieve, and then perform secondary filtration separation with a 40 μm cell filter membrane. The filtrate is the filtrate containing stem cells;

[0092] (3) Expansion culture of stem cells: Transfer the finally filtered stem cell-containing filtrate into Erlenmeyer flasks respectively, and add MS medium containing amphotericin B at a concentration of 0.9 mg / L, double antibiotics at a mass concentration of 0.8%, 6-BA at 0.9 mg / L, and NAA at 0.9 mg / L. Divide the Erlenmeyer flasks into three groups, and add lanthanum nitrate at a concentration of 300 mg / L to the medium in each group. Place the Erlenmeyer flasks on a vertical constant temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C for shaking culture for 2 days. After the culture is completed, transfer the stem cell-containing medium from 1 Erlenmeyer flask evenly to 3 Erlenmeyer flasks respectively, and continue to add MS medium containing amphotericin B at a concentration of 0.9 mg / L, double antibiotics at a mass concentration of 0.8%, 6-BA at 0.9 mg / L, and NAA at 0.9 mg / L. Then divide the Erlenmeyer flasks into three groups, and add lanthanum nitrate at a concentration of 300 mg / L to the medium in each group until the amount of the medium in each Erlenmeyer flask reaches about 1 / 3 position. Place it on a vertical constant temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C for subculture;

[0093] (4) Collection of stem cells: Expand and culture the stem cells for 48 h and collect them respectively. Pour the stem cell-containing medium into a centrifuge bottle, balance it, and place it in a centrifuge with a rotation speed of 2200 r / min and a centrifugation time of 10 minutes. After centrifugation, discard the supernatant to obtain the stem cells of Dendrobium officinale. Freeze-dry or microwave-dry the obtained stem cells. After complete drying, use a small pulverizer to pulverize and sieve to obtain the Dendrobium officinale stem cell powder.

[0094] Example 6

[0095] The orchid plant used in this example is Dendrobium officinale, and it relates to a method for culturing Dendrobium officinale stem cells. The specific steps are as follows:

[0096] (1) Pretreatment of young stems: First, take the young stems of Dendrobium officinale and rinse them with clean water for 3 h, and carefully brush the parts that are not easy to clean with a brush to remove dust and other attachments. Then wash and disinfect them with an aqueous alcohol solution with a volume concentration of 75% for 20 s, and then rinse them with sterile water 3 times. Next, disinfect them with a disinfectant containing sodium hypochlorite for 5 min, and finally rinse them with sterile water 3 times for standby;

[0097] (2) Obtaining of stem cells: Under a biosafety cabinet, cut off the shoot tip of the disinfected young stem in step (1) with a surgical blade, strip the shoot tip layer by layer with a dissecting needle and forceps, leaving 5 - 6 leaf primordia, then cut it at 0.5 mm from the shoot tip with a surgical blade. In a sufficient interval space, pick out the shoot tip with Dendrobium officinale Kimura et Migo meristem with a dissecting needle, and then transfer the isolated shoot tip meristem into 3 Erlenmeyer flasks containing culture medium for cultivation. The culture medium is MS medium containing amphotericin B at a concentration of 1.2 mg / L, double antibody at a mass concentration of 1.3%, 6 - BA at 1.1 mg / L, and NAA at 1.2 mg / L. Additionally, lanthanum nitrate at a concentration of 300 mg / L is added to the culture medium respectively, the pH value of the culture medium is 6.2. Place the Erlenmeyer flasks on a vertical constant temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C, and perform shaking culture for 3 days. After the cultivation is completed, first filter out larger flakes and debris with a No. 2 sieve, and then perform secondary filtration separation with a 40 - um cell filter membrane. The filtrate is the filtrate containing stem cells;

[0098] (3) Sub - culture expansion of stem cells: Transfer the finally filtered filtrate containing stem cells into Erlenmeyer flasks respectively, and add MS medium containing amphotericin B at a concentration of 1.2 mg / L, double antibody at a mass concentration of 1.3%, 6 - BA at 1.1 mg / L, and NAA at 1.2 mg / L. Divide the Erlenmeyer flasks into three groups, and add lanthanum nitrate at a concentration of 300 mg / L to the culture medium of each group respectively. Place the Erlenmeyer flasks on a vertical constant temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C, and perform shaking culture for 2 days. After the cultivation is completed, transfer the culture medium containing stem cells from 1 Erlenmeyer flask to 3 Erlenmeyer flasks evenly, and continue to add MS medium containing amphotericin B at a concentration of 1.2 mg / L, double antibody at a mass concentration of 1.3%, 6 - BA at 1.1 mg / L, and NAA at 1.2 mg / L to it. Then divide the Erlenmeyer flasks into three groups, and add lanthanum nitrate at a concentration of 300 mg / L to the culture medium of each group respectively until the amount of the culture medium in each Erlenmeyer flask reaches about 1 / 3 of the position. Place it on a vertical constant temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C for sub - culture;

[0099] (4) Collection of stem cells: Expand and culture the stem cells for 48 h and collect them respectively. Pour the culture medium containing stem cells into a centrifuge bottle, balance it, and place it in a centrifuge with a rotation speed of 2200 r / min and a centrifugation time of 10 minutes. After the centrifugation is completed, discard the supernatant, and Dendrobium officinale Kimura et Migo stem cells are obtained. Freeze - dry or microwave - dry the obtained stem cells. After complete drying, use a small crusher to crush and sieve them to obtain Dendrobium officinale Kimura et Migo stem cell powder.

[0100] Experimental Example 2

[0101] The stem cells obtained in Examples 1-6 were weighed and measured. Five experiments were conducted and the average value was taken. The results are shown in Tables 2 and 3.

[0102] Table 2 Comparison results of the weights of Dendrobium officinale stem cells using B5 medium

[0103] Example 1 Example 3 Example 4 Added lanthanum nitrate concentration / mg / L 300 300 300 Added component concentration Medium Low High Culture medium type B5 B5 B5 6-BA concentration / mg / L 0.100 0.099 0.130 Sucrose concentration / mg / L 15 12 17 Methyl jasmonate concentration / mg / L 0.50 0.49 0.54 Triacontanol concentration / mg / L 0.100 0.098 0.120 Average weight / g 94.56 94.01 93.87

[0104] Table 3 Comparison results of the weights of Dendrobium officinale stem cells using MS medium

[0105] Example 2 Example 5 Example 6 Added lanthanum nitrate concentration / mg / L 300 300 300 Added component concentration Medium Low High Culture medium type MS MS MS Amphotericin B concentration / mg / L 1.0 0.9 1.2 Dual antibiotic concentration / % 1.0 0.8 1.3 6-BA concentration / mg / L 1.0 0.9 1.1 NAA concentration / mg / L 1.0 0.9 1.2 Average weight / g 122.13 121.33 121.75

[0106] As can be seen from Tables 2 and 3 above, when the concentrations of the relevant components added to the B5 medium and the MS medium are within the scope of the present invention's scheme, they all have a certain effective effect, which is beneficial to the culture of stem cells.

[0107] Example 7

[0108] This example takes Dendrobium loddigesii as an example and relates to the method for culturing and collecting Dendrobium loddigesii stem cells under the condition of B5 medium added with lanthanum nitrate. The specific steps are as follows:

[0109] (1) Pretreatment of tender stems: First, take the tender stems of Dendrobium loddigesii and rinse them with clean water for 3 h, and carefully brush the parts that are not easy to clean with a brush to remove dust and other attachments. Then, clean and disinfect them with an aqueous alcohol solution with a volume concentration of 75% for 20 s, and then rinse them 3 times with sterile water. Next, disinfect them with a disinfectant containing sodium hypochlorite for 5 min, and finally rinse them 3 times with sterile water for standby;

[0110] (2) Obtaining of stem cells: Cut off the stem tips of the disinfected tender stems in step (1) with a surgical blade in a biosafety cabinet, and layer by layer peel the stem tips with a dissecting needle and forceps, leaving 5-6 leaf primordia. Then, cut at 0.5 mm from the stem tip with a surgical blade. In a sufficient interval space, the stem tips with the meristem of Dendrobium loddigesii can be picked out with a dissecting needle. Then, put the separated stem tip meristems into a triangular flask containing the medium for culture. The medium is B5 medium containing 0.100 mg / L of 6-BA, 15 mg / L of sucrose, 0.50 mg / L of methyl jasmonate, and 0.100 mg / L of triacontanol. The medium also contains lanthanum nitrate with a final concentration of 300 mg / L, and the pH value of the medium is 6.2; Place it on a vertical constant temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C for shaking culture for 3 days; After the culture is completed, first filter out the larger flakes and fragments with a No. 2 sieve, and then perform secondary filtration separation with a 40-μm cell filter membrane. The filtrate is the filtrate containing stem cells;

[0111] (3) Expansion culture of stem cells: Transfer the filtered filtrate containing stem cells into an Erlenmeyer flask, and add B5 medium containing 0.100 mg / L of 6-BA, 15 mg / L of sucrose, 0.50 mg / L of methyl jasmonate, and 0.100 mg / L of triacontanol. Additionally, add lanthanum nitrate with a final concentration of 300 mg / L to the medium. Place the Erlenmeyer flask on a vertical constant-temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C for shaking culture for 2 days. After the culture is completed, transfer the medium containing stem cells evenly from 1 Erlenmeyer flask to 3 Erlenmeyer flasks, and continue to add B5 medium containing 0.100 mg / L of 6-BA, 15 mg / L of sucrose, 0.50 mg / L of methyl jasmonate, and 0.100 mg / L of triacontanol (lanthanum nitrate with a final concentration of 300 mg / L is added to the medium) until the amount of medium in each Erlenmeyer flask reaches about 1 / 3 of the position. Then, place it on a vertical constant-temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C for subculture;

[0112] (4) Collection of stem cells: After 48 h of subculture of stem cells, collect them. Pour the medium containing stem cells into a centrifuge bottle, balance it, and then place it in a centrifuge with a rotation speed of 2200 r / min for 10 minutes. After centrifugation, discard the supernatant to obtain the stem cells of Dendrobium loddigesii. Freeze-dry or microwave-dry the obtained stem cells. After complete drying, use a small pulverizer to pulverize and sieve them to obtain the Dendrobium loddigesii stem cell powder.

[0113] Example 8

[0114] This example takes Dendrobium loddigesii as an example and involves the culture and collection methods of Dendrobium loddigesii stem cells under the condition of MS medium added with lanthanum nitrate. The specific steps are as follows:

[0115] (1) Pretreatment of tender stems: First, take the tender stems of Dendrobium loddigesii, rinse them with clean water for 3 h, and carefully brush the parts that are not easy to clean with a brush to remove dust and other attachments. Then, wash and disinfect them with an aqueous alcohol solution with a volume concentration of 75% for 20 s, and then rinse them 3 times with sterile water. Next, disinfect them with a disinfectant containing sodium hypochlorite for 5 min, and finally rinse them 3 times with sterile water for standby;

[0116] (2) Obtaining stem cells: After disinfecting the tender stems in step (1), cut off the shoot tips with a surgical blade in a biosafety cabinet. Use a dissecting needle and forceps to strip the shoot tips layer by layer, leaving 5 - 6 leaf primordia. Then cut at 0.5 mm from the shoot tip with a surgical blade. In a sufficient space, pick out the shoot tips with the Dendrobium loddigesii Rolfe meristem with a dissecting needle. Then transfer the isolated shoot tip meristems into 3 Erlenmeyer flasks containing culture medium. The culture medium is MS medium containing amphotericin B at a concentration of 1.0 mg / L, double antibody (100X penicillin - streptomycin mixture) at a volume concentration of 1.0%, 1.0 mg / L of 6 - BA, 1.0 mg / L of NAA. Lanthanum nitrate with concentration gradients of 100 mg / L, 200 mg / L, and 300 mg / L is also added to the medium respectively. Place the Erlenmeyer flasks in a vertical constant temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C for shaking culture for 3 days. After the culture, first filter out larger flakes and debris with a No. 2 sieve, and then perform secondary filtration separation with a 40 - μm cell filter membrane. The filtrate is the filtrate of stem cells;

[0117] (3) Expansion culture of stem cells: Transfer the finally filtered filtrate containing stem cells into an Erlenmeyer flask, and add MS medium containing amphotericin B at a concentration of 1.0 mg / L, double antibody at a volume concentration of 1.0%, 1.0 mg / L of 6 - BA, 1.0 mg / L of NAA. Lanthanum nitrate with concentration gradients of 100 mg / L, 200 mg / L, and 300 mg / L is also added to the medium respectively. Place the Erlenmeyer flask in a vertical constant temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C for shaking culture for 2 days. After the culture, evenly transfer the culture medium containing stem cells from 1 Erlenmeyer flask to 3 Erlenmeyer flasks, and continue to add MS medium (containing amphotericin B at a concentration of 1.0 mg / L, double antibody at a volume concentration of 1.0%, 1.0 mg / L of 6 - BA, 1.0 mg / L of NAA, and lanthanum nitrate with concentration gradients of 100 mg / L, 200 mg / L, and 300 mg / L respectively) to each Erlenmeyer flask until the amount of medium in each flask reaches about 1 / 3 of the position. Then perform sub - culture in a vertical constant temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C;

[0118] (4) Collection of stem cells: The stem cells were subcultured for 48 h and then collected. The culture medium containing the stem cells was poured into a centrifuge bottle. After balancing, it was placed in a centrifuge at a rotational speed of 2200 r / min for 10 minutes. After centrifugation, the supernatant was discarded, and the stem cells of a certain plant were obtained. The obtained stem cells were freeze-dried or microwave-dried. After complete drying, they were crushed and sieved using a small crusher to obtain Dendrobium loddigesii Rolfe stem cell powder. Collect the Dendrobium loddigesii Rolfe stem cells obtained from the culture media with different concentrations of lanthanum nitrate.

[0119] Experimental Example 3

[0120] This experimental example involves an experiment on the effect of lanthanum nitrate on Dendrobium loddigesii Rolfe stem cells.

[0121] Set Comparative Example 3: No lanthanum nitrate was added to the B5 medium, and the others were the same as in Example 7.

[0122] Comparative Example 4: No lanthanum nitrate was added to the MS medium, and the others were the same as in Example 8.

[0123] The finally obtained Dendrobium loddigesii Rolfe stem cells in Examples 7 and 8 and Comparative Examples 3 and 4 were weighed separately. Each group was experimented 5 times, and the average value was finally taken. The results are shown in Table 4.

[0124] Table 4 Comparison results of the weights of Dendrobium loddigesii Rolfe stem cells

[0125] Example 7 Comparative Example 3 Example 8 Example 8 Example 8 Comparative Example 4 Lanthanum nitrate concentration / mg / L 300 0 100 200 300 0 Culture medium type B5 B5 MS MS MS MS Weight / g 88.12 78.56 103.56 111.44 120.13 87.43

[0126] It can be seen from Table 4 that the weights of the Dendrobium loddigesii Rolfe stem cells obtained in Examples 7 and 8 are greater than those in Comparative Examples 3 and 4, indicating that the culture medium containing lanthanum nitrate at a concentration of 100 - 300 mg / L can culture a large number of stem cells in a shorter time. The weights of the stem cells obtained from the culture media with different concentrations of lanthanum nitrate in Example 8 are significantly better than those in Example 7, indicating that the composition and ratio of the culture medium in Example 8 are more suitable for culturing a large number of Dendrobium loddigesii Rolfe stem cells in a short time.

[0127] Example 9

[0128] This example takes Dendrobium fimbriatum Hook. as an example and involves the method for culturing and collecting Dendrobium fimbriatum Hook. stem cells under the condition of a B5 medium added with lanthanum nitrate. The specific steps are as follows:

[0129] (1) Pretreatment of young stems: First, take the young stems of Dendrobium fimbriatum Hook., rinse them with clear water for 3 h, and carefully brush the parts that are not easy to clean with a brush to remove dust and other attachments. Then, clean and disinfect them with an aqueous alcohol solution with a volume concentration of 75% for 20 s, and then rinse them 3 times with sterile water. Next, disinfect them with a disinfectant containing sodium hypochlorite for 5 min, and finally rinse them 3 times with sterile water for standby;

[0130] (2) Obtaining stem cells: The stem tip of the tender stem disinfected in step (1) was cut off with a surgical blade in a biosafety cabinet, and the stem tip was peeled off layer by layer with a dissecting needle and tweezers, leaving 5-6 leaf primordia, and then cut off with a surgical blade at 0.5 mm from the stem tip. The stem tip with the meristem of Dendrobium officinale can be picked out with a dissecting needle within a sufficient spacing, and then the separated stem tip was placed in a triangular flask containing a culture medium for culture, wherein the culture medium contained 0.100 mg / L 6-BA, 15 mg / L sucrose, 0 .50mg / L methyl jasmonate and 0.100mg / L triacontanol B5 medium, 300mg / L lanthanum nitrate is also added to the medium, the pH value of the medium is 6.2, the conical flask is placed in a vertical constant temperature oscillator, the speed is 110r / min, the temperature is 22°C, and the shaking table culture is carried out for 3 days; after the culture is completed, the larger flakes and fragments are first filtered out with a No. 2 sieve, and then a 40um cell filter membrane is used for secondary filtration and separation, and the filtrate is the filtrate containing stem cells;

[0131] (3) Expansion culture of stem cells: The filtrate containing stem cells obtained by the final filtration was transferred to a conical flask, and B5 medium containing 0.100 mg / L 6-BA, 15 mg / L sucrose, 0.50 mg / L methyl jasmonate and 0.100 mg / L triacontanol was added. In addition, 300 mg / L lanthanum nitrate was added to the medium, and the medium was placed in a vertical constant temperature oscillator at a speed of 110 r / min and a temperature of 22° C. for shaking culture. The culture time was 2 days. After the culture was completed, the medium was The culture medium containing stem cells was evenly transferred from one flask to three flasks, and B5 culture medium (with lanthanum nitrate at a concentration of 300 mg / L added to the culture medium) containing 0.100 mg / L 6-BA, 15 mg / L sucrose, 0.50 mg / L methyl jasmonate and 0.100 mg / L triacontanol was added until the amount of the culture medium in each flask reached about 1 / 3, and the culture was performed in a vertical constant temperature oscillator at a speed of 110 r / min and a temperature of 22° C. for subculture;

[0132] (4) Collecting stem cells: The stem cells are subcultured for 48 hours and then collected. The culture medium containing the stem cells is poured into a centrifuge bottle, which is then balanced and placed in a centrifuge at a speed of 2200 r / min for 10 minutes. After the centrifugation, the supernatant is discarded to obtain the stem cells of Dendrobium officinale. The obtained stem cells are freeze-dried or microwave-dried. After complete drying, they are crushed and sieved using a small grinder to obtain Dendrobium officinale stem cell powder.

[0133] Example 10

[0134] This example takes Dendrobium fimbriatum Hook. var. oculatum Hook. as an example and involves a method for culturing and collecting stem cells of Dendrobium fimbriatum Hook. var. oculatum Hook. under the condition of MS medium added with lanthanum nitrate. The specific steps are as follows:

[0135] (1) Pretreatment of young stems: First, take the young stems of Dendrobium fimbriatum Hook. var. oculatum Hook. and rinse them with clean water for 3 h, and carefully brush the parts that are not easy to clean with a brush to remove dust and other attachments. Then, clean and disinfect them with an aqueous alcohol solution with a volume concentration of 75% for 20 s, and then rinse them 3 times with sterile water. Next, disinfect them with a disinfectant containing sodium hypochlorite for 5 min, and finally rinse them 3 times with sterile water for standby;

[0136] (2) Obtaining of stem cells: Cut off the stem tips of the disinfected young stems in step (1) with a surgical blade in a biosafety cabinet, and layer by layer peel the stem tips with a dissecting needle and forceps, leaving 5 - 6 leaf primordia. Then, cut at 0.5 mm from the stem tip with a surgical blade. In a sufficient interval space, the stem tips with the meristem of Dendrobium fimbriatum Hook. var. oculatum Hook. can be picked out with a dissecting needle. Then, put the separated stem tips into 3 triangular flasks containing the culture medium for culture. The culture medium is MS medium containing amphotericin B with a concentration of 1.0 mg / L, double antibody (100X penicillin-streptomycin mixture) with a volume concentration of 1.0%, 1.0 mg / L of 6-BA, and 1.0 mg / L of NAA. In addition, lanthanum nitrate with concentration gradients of 100 mg / L, 200 mg / L, and 300 mg / L is added to the culture medium respectively. Place the triangular flasks in a vertical constant temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C for shaking culture for 3 days. After the culture is completed, first filter out the larger flakes and fragments with a No. 2 sieve, and then perform secondary filtration separation with a 40-μm cell filter membrane. The filtrate is the filtrate containing stem cells;

[0137] (3) Expansion culture of stem cells: Transfer the finally filtered filtrate containing stem cells into an Erlenmeyer flask, and add MS medium containing amphotericin B at a concentration of 1.0 mg / L, double antibiotics at a volume concentration of 1.0%, 6-BA at 1.0 mg / L, and NAA at 1.0 mg / L. Additionally, lanthanum nitrate at concentrations of 100 mg / L, 200 mg / L, and 300 mg / L is added respectively, and it is placed in a vertical constant temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C for shaking culture for 2 days. After the culture is completed, transfer the medium containing stem cells evenly from 1 Erlenmeyer flask to 3 Erlenmeyer flasks, and continue to add MS medium containing amphotericin B at a concentration of 1.0 mg / L, double antibiotics at a volume concentration of 1.0%, 6-BA at 1.0 mg / L, and NAA at 1.0 mg / L (lanthanum nitrate at concentrations of 100 mg / L, 200 mg / L, and 300 mg / L is added to the medium respectively) until the amount of medium in each Erlenmeyer flask reaches about 1 / 3 position, and perform subculture in a vertical constant temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C;

[0138] (4) Collection of stem cells: After the stem cells are subcultured for 48 h, collect them. Pour the medium containing stem cells into a centrifuge bottle, balance it, and then place it in a centrifuge with a rotation speed of 2200 r / min and a centrifugation time of 10 minutes. After centrifugation, discard the supernatant to obtain the stem cells of Dendrobium fimbriatum Hook. var. oculatum Hook.; Freeze-dry or microwave-dry the obtained stem cells. After complete drying, use a small crusher to crush and sieve them to obtain the stem cell powder of Dendrobium fimbriatum Hook. var. oculatum Hook.; Collect the stem cell powder of Dendrobium fimbriatum Hook. var. oculatum Hook. obtained from media with different concentrations of lanthanum nitrate.

[0139] Test Example 4

[0140] This test example involves an experiment on the effect of lanthanum nitrate on the stem cells of Dendrobium fimbriatum Hook. var. oculatum Hook.

[0141] Set Comparative Example 5: No lanthanum nitrate is added to the B5 medium, and the others are the same as in Example 9.

[0142] Comparative Example 6: No lanthanum nitrate is added to the MS medium, and the others are the same as in Example 10.

[0143] Weigh the finally obtained stem cells of Dendrobium fimbriatum Hook. var. oculatum Hook. in the above Examples 9 and 10 and Comparative Examples 5 and 6 respectively. Conduct 5 experiments for each group and finally take the average value. The results are shown in Table 5.

[0144] Table 5 Comparison results of the weights of stem cells of Dendrobium fimbriatum Hook. var. oculatum Hook.

[0145] Example 9 Comparative Example 5 Example 10 Example 10 Example 10 Comparative Example 6 Lanthanum nitrate concentration / mg / L 300 0 100 200 300 0 Culture medium type B5 B5 MS MS MS MS Weight / g 94.13 84.56 113.56 117.44 126.53 76.43

[0146] As can be seen from Table 3, the weight ratio of the Dendrobium fimbriatum Hook. var. oculatum Hook. stem cells obtained in Examples 9 and 10 is greater than that in Comparative Examples 5 and 6, indicating that the culture medium supplemented with lanthanum nitrate at a concentration of 100-300 mg / L can cultivate a large number of stem cells in a relatively short time. The weight of the stem cells obtained from the culture medium with different concentrations of lanthanum nitrate in Example 10 is significantly better than that in Example 9, indicating that the composition and ratio of the culture medium in Example 10 are more suitable for cultivating a large number of Dendrobium fimbriatum Hook. var. oculatum Hook. stem cells in a short time.

[0147] Example 11

[0148] This example takes Dendrobium chrysanthum Wall. as an example and relates to a method for culturing and collecting Dendrobium chrysanthum Wall. stem cells under the condition of B5 culture medium supplemented with lanthanum nitrate. The specific steps are as follows:

[0149] (1) Pretreatment of young stems: First, take the young stems of Dendrobium chrysanthum Wall. and rinse them with clean water for 3 h, and carefully brush the parts that are not easy to clean with a brush to remove dust and other attachments. Then, clean and disinfect them with 75% (v / v) alcohol for 20 s, and then rinse them 3 times with sterile water. Next, disinfect them with a disinfectant containing sodium hypochlorite for 5 min, and finally rinse them 3 times with sterile water for standby;

[0150] (2) Obtaining of stem cells: Cut off the shoot tips of the disinfected young stems in step (1) with a surgical blade in a biosafety cabinet, and layer by layer peel the shoot tips with a dissecting needle and forceps, leaving 5-6 leaf primordia. Then, cut at 0.5 mm from the shoot tip with a surgical blade. In a sufficient interval space, the shoot tips with the meristem of Dendrobium chrysanthum Wall. can be picked out with a dissecting needle. Then, put the separated shoot tips into a triangular flask containing the culture medium for culture. The culture medium is B5 culture medium containing 0.100 mg / L of 6-BA, 15 mg / L of sucrose, 0.50 mg / L of methyl jasmonate and 0.100 mg / L of triacontanol. The culture medium is also supplemented with lanthanum nitrate at a concentration of 300 mg / L. The pH value of the culture medium is 6.2. Place it on a vertical constant temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C for shaking culture for 3 days. After the culture is completed, first filter out the larger flakes and fragments with a No. 2 sieve, and then perform secondary filtration separation with a 40-μm cell filter membrane. The filtrate is the filtrate containing stem cells;

[0151] (3) Expansion culture of stem cells: Transfer the finally filtered filtrate containing stem cells into an Erlenmeyer flask, and add B5 medium containing 0.100 mg / L of 6-BA, 15 mg / L of sucrose, 0.50 mg / L of methyl jasmonate, and 0.100 mg / L of triacontanol. Additionally, add lanthanum nitrate with a concentration of 300 mg / L to the medium, and place it in a vertical constant temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C for shaking culture. The culture time is 2 days. After the culture is completed, transfer the medium containing stem cells evenly from 1 Erlenmeyer flask to 3 Erlenmeyer flasks, and continue to add B5 medium containing 0.100 mg / L of 6-BA, 15 mg / L of sucrose, 0.50 mg / L of methyl jasmonate, and 0.100 mg / L of triacontanol (lanthanum nitrate with a concentration of 300 mg / L is also added to the medium) until the amount of medium in each Erlenmeyer flask reaches about 1 / 3 of the position. Then, perform subculture in a vertical constant temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C;

[0152] (4) Collection of stem cells: The stem cells are collected after 48 h of subculture. Pour the medium containing stem cells into a centrifuge bottle, balance it, and then place it in a centrifuge with a rotation speed of 2200 r / min and a centrifugation time of 10 minutes. After centrifugation, discard the supernatant to obtain the stem cells of Dendrobium officinale. Freeze-dry or microwave-dry the obtained stem cells. After complete drying, use a small pulverizer to pulverize and sieve them to obtain the Dendrobium officinale stem cell powder.

[0153] Example 12

[0154] This example takes Dendrobium officinale as an example and relates to the culture and collection methods of Dendrobium officinale stem cells under the condition of MS medium added with lanthanum nitrate. The specific steps are as follows:

[0155] (1) Pretreatment of young stems: First, take the young stems of Dendrobium officinale and rinse them with clean water for 3 h, and carefully brush the parts that are not easy to clean with a brush to remove dust and other attachments. Then, wash and disinfect them with an aqueous alcohol solution with a volume concentration of 75% for 20 s, and then rinse them with sterile water 3 times. Next, disinfect them with a disinfectant containing sodium hypochlorite for 5 min, and finally rinse them with sterile water 3 times for standby;

[0156] (2) Obtaining stem cells: After disinfecting the tender stems in step (1), cut off the shoot tips with a surgical blade in a biosafety cabinet. Use a dissecting needle and forceps to strip the shoot tips layer by layer, leaving 5 - 6 leaf primordia. Then cut at 0.5 mm from the shoot tip with a surgical blade. In a sufficient interval space, pick out the shoot tips with the Dendrobium officinale Kimura et Migo meristem with a dissecting needle. Then transfer the separated shoot tips into 3 Erlenmeyer flasks containing culture medium for cultivation. The culture medium is an MS medium containing amphotericin B at a concentration of 1.0 mg / L, double antibody (100X penicillin-streptomycin mixture) at a volume concentration of 1.0%, 1.0 mg / L of 6-BA, and 1.0 mg / L of NAA. Additionally, lanthanum nitrate at concentrations of 100 mg / L, 200 mg / L, and 300 mg / L is added to the culture medium respectively. Place the Erlenmeyer flasks in a vertical constant temperature oscillator at a rotation speed of 110 r / min and a temperature of 22°C for shaking culture for 3 days. After the cultivation, first filter out the larger flakes and debris with a No. 2 sieve, and then perform secondary filtration separation with a 40-μm cell filter membrane. The filtrate is the filtrate containing stem cells;

[0157] (3) Subculture of stem cells: Transfer the finally filtered filtrate containing stem cells into an Erlenmeyer flask, and add an MS medium containing amphotericin B at a concentration of 1.0 mg / L, double antibody at a volume concentration of 1.0%, 1.0 mg / L of 6-BA, and 1.0 mg / L of NAA. Additionally, lanthanum nitrate at concentrations of 100 mg / L, 200 mg / L, and 300 mg / L is added to the culture medium respectively. Place the Erlenmeyer flask in a vertical constant temperature oscillator at a rotation speed of 110 r / min and a temperature of 22°C for shaking culture for 2 days. After the cultivation, transfer the culture medium containing stem cells from 1 Erlenmeyer flask evenly into 3 Erlenmeyer flasks, and continue to add an MS medium containing amphotericin B at a concentration of 1.0 mg / L, double antibody at a volume concentration of 1.0%, 1.0 mg / L of 6-BA, and 1.0 mg / L of NAA (the culture medium also contains lanthanum nitrate at concentrations of 100 mg / L, 200 mg / L, and 300 mg / L respectively) to each Erlenmeyer flask until the amount of the culture medium in each Erlenmeyer flask reaches about 1 / 3 of the position. Place it in a vertical constant temperature oscillator at a rotation speed of 110 r / min and a temperature of 22°C for subculture;

[0158] (4) Collection of stem cells: The stem cells were subcultured for 48 hours and then collected. The culture medium containing the stem cells was poured into a centrifuge bottle, balanced, placed in a centrifuge, centrifuged at a speed of 2200 r / min for 10 minutes. After centrifugation, the supernatant was discarded to obtain the stem cells of Dendrobium chrysotoxum. The obtained stem cells were freeze-dried or microwave-dried. After complete drying, they were crushed and sieved using a small crusher to obtain the stem cell powder of Dendrobium chrysotoxum. The stem cell powder of Dendrobium chrysotoxum obtained from the culture media with different concentrations of lanthanum nitrate was collected.

[0159] Experimental Example 5

[0160] This experimental example involves an experiment on the effect of lanthanum nitrate on the stem cells of Dendrobium fimbriatum.

[0161] Set Comparative Example 7: Lanthanum nitrate was not added to the B5 medium, and the others were the same as in Example 11.

[0162] Set Comparative Example 8: Lanthanum nitrate was not added to the MS medium, and the others were the same as in Example 12.

[0163] The finally obtained stem cells of Dendrobium chrysotoxum in Examples 11 and 12 and Comparative Examples 7 and 8 were weighed separately. Each group was experimented 5 times, and the average value was finally taken. The results are shown in Table 6.

[0164] Table 6 Comparison results of the weights of the stem cells of Dendrobium chrysotoxum

[0165] Example 11 Comparative Example 7 Example 12 Example 12 Example 12 Comparative Example 8 Lanthanum nitrate concentration / mg / L 300 0 100 200 300 0 Culture medium type B5 B5 MS MS MS MS Weight / g 77.13 70.56 102.56 110.44 116.13 81.43

[0166] It can be seen from Table 6 that the weights of the stem cells of Dendrobium chrysotoxum obtained in Examples 11 and 12 are greater than those in Comparative Examples 7 and 8, indicating that the culture media containing lanthanum nitrate at a concentration of 100 - 300 mg / L can cultivate a large number of stem cells in a relatively short time. The weights of the stem cells obtained from the culture media with different concentrations of lanthanum nitrate in Example 12 are significantly better than those in Example 11, indicating that the composition and ratio of the culture medium in Example 12 are more suitable for cultivating a large number of stem cells of Dendrobium chrysotoxum in a short time.

[0167] Example 13

[0168] This example takes Dendrobium nobile as an example and involves the cultivation and collection methods of the stem cells of Dendrobium nobile under the condition of adding lanthanum nitrate to the B5 medium. The specific steps are as follows:

[0169] (1) Pretreatment of young stems: First, take the young stems of Dendrobium nobile, rinse them with clean water for 3 hours, carefully brush the parts that are not easy to clean with a brush to remove dust and other attachments, then wash and disinfect them with an aqueous alcohol solution with a volume concentration of 75% for 20 seconds, then rinse them 3 times with sterile water, next disinfect them with a disinfectant containing sodium hypochlorite for 5 minutes, and finally rinse them 3 times with sterile water for standby;

[0170] (2) Obtaining stem cells: After disinfecting the tender stems in step (1), cut off the shoot tips with a surgical blade in a biosafety cabinet. Use a dissecting needle and forceps to peel the shoot tips layer by layer, leaving 5 - 6 leaf primordia. Then cut at 0.5 mm from the shoot tip with a surgical blade. In a sufficient space, pick out the shoot tips with the meristem of Dendrobium nobile Lindl. using a dissecting needle. Then transfer the separated shoot tips into a triangular flask containing the culture medium for cultivation. The culture medium is B5 medium containing 0.100 mg / L of 6 - BA, 15 mg / L of sucrose, 0.50 mg / L of methyl jasmonate, and 0.100 mg / L of triacontanol. Additionally, lanthanum nitrate with a final concentration of 300 mg / L is added to the medium. The pH value of the medium is 6.2. Place the triangular flask on a vertical constant temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C for shaking culture for 3 days. After the cultivation, first filter out larger flakes and fragments with a No. 2 sieve, and then perform secondary filtration separation with a 40 - μm cell filter membrane. The filtrate is the filtrate containing stem cells;

[0171] (3) Sub - culture expansion of stem cells: Transfer the finally filtered filtrate containing stem cells into a triangular flask, and add B5 medium containing 0.100 mg / L of 6 - BA, 15 mg / L of sucrose, 0.50 mg / L of methyl jasmonate, and 0.100 mg / L of triacontanol. Additionally, lanthanum nitrate with a concentration of 300 mg / L is added to the medium. Place it on a vertical constant temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C for shaking culture for 2 days. After the cultivation, evenly transfer the medium containing stem cells from 1 triangular flask to 3 triangular flasks, and continue to add B5 medium containing 0.100 mg / L of 6 - BA, 15 mg / L of sucrose, 0.50 mg / L of methyl jasmonate, and 0.100 mg / L of triacontanol (lanthanum nitrate with a concentration of 300 mg / L is also added to the medium) until the amount of the medium in each triangular flask reaches about 1 / 3 of the position. Then perform sub - culture on a vertical constant temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C;

[0172] (4) Collecting stem cells: Perform sub - culture of stem cells for 48 h and then collect. Pour the medium containing stem cells into a centrifuge bottle, balance it, and place it in a centrifuge with a rotation speed of 2200 r / min for 10 minutes. After centrifugation, discard the supernatant to obtain the stem cells of Dendrobium nobile Lindl. Freeze - dry or microwave - dry the obtained stem cells. After complete drying, use a small pulverizer to pulverize and sieve to obtain the stem cell powder of Dendrobium nobile Lindl.

[0173] Example 14

[0174] This example takes *Dendrobium nobile* as an example and involves a method for culturing and collecting stem cells of *Dendrobium nobile* under the condition of MS medium added with lanthanum nitrate. The specific steps are as follows:

[0175] (1) Pretreatment of tender stems: First, take the tender stems of *Dendrobium nobile*, rinse them with clean water for 3 h, and carefully brush the parts that are not easy to clean with a brush to remove dust and other attachments. Then, clean and disinfect them with an aqueous alcohol solution with a volume concentration of 75% for 20 s, and then rinse them 3 times with sterile water. Next, disinfect them with a disinfectant containing sodium hypochlorite for 5 min, and finally rinse them 3 times with sterile water for standby;

[0176] (2) Obtaining of stem cells: Cut off the stem tips of the disinfected tender stems in step (1) with a surgical blade in a biosafety cabinet, layer by layer peel the stem tips with a dissecting needle and forceps, leaving 5 - 6 leaf primordia, and then cut at 0.5 mm from the stem tip with a surgical blade. In a sufficient interval space, the stem tips with the meristem of *Dendrobium nobile* can be picked out with a dissecting needle. Then, put the separated stem tips into 3 Erlenmeyer flasks containing the medium for culture. The medium is MS medium containing amphotericin B with a concentration of 1.0 mg / L, double antibody (100X penicillin-streptomycin mixture) with a volume concentration of 1.0%, 1.0 mg / L of 6-BA, and 1.0 mg / L of NAA. Lanthanum nitrate with concentrations of 100 mg / L, 200 mg / L, and 300 mg / L is respectively added to the 3 media. Place the Erlenmeyer flasks in a vertical constant temperature oscillator, with a rotation speed of 110 r / min and a temperature of 22 °C, and perform shaking culture for 3 days. After the culture is completed, first filter out larger flakes and fragments with a No. 2 sieve, and then perform secondary filtration separation with a 40-μm cell filter membrane. The filtrate is the filtrate of stem cells;

[0177] (3) Expansion culture of stem cells: Transfer the finally filtered filtrate containing stem cells into an Erlenmeyer flask, and add MS medium containing amphotericin B at a concentration of 1.0 mg / L, double antibiotics at a volume concentration of 1.0%, 6-BA at a concentration of 1.0 mg / L, and NAA at a concentration of 1.0 mg / L. Lanthanum nitrate at concentrations of 100 mg / L, 200 mg / L, and 300 mg / L is also added to the 3 media respectively. Place the Erlenmeyer flask on a vertical constant temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C for shaking culture for 2 days. After the culture, transfer the medium containing stem cells from 1 Erlenmeyer flask evenly into 3 Erlenmeyer flasks, and continue to add MS medium containing amphotericin B at a concentration of 1.0 mg / L, double antibiotics at a volume concentration of 1.0%, 6-BA at a concentration of 1.0 mg / L, and NAA at a concentration of 1.0 mg / L (lanthanum nitrate at concentrations of 100 mg / L, 200 mg / L, and 300 mg / L is added to the other 3 media respectively) until the amount of medium in each Erlenmeyer flask reaches about 1 / 3 of the position. Then, perform subculture on a vertical constant temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C;

[0178] (4) Collection of stem cells: After 48 h of subculture of stem cells, collect them. Pour the medium containing stem cells into a centrifuge bottle, balance it, and then place it in a centrifuge with a rotation speed of 2200 r / min for 10 minutes. After centrifugation, discard the supernatant to obtain the stem cells of Dendrobium nobile Lindl. Freeze-dry or microwave-dry the obtained stem cells. After complete drying, use a small pulverizer to pulverize and sieve them to obtain the stem cell powder of Dendrobium nobile Lindl. Collect the stem cell powder of Dendrobium nobile Lindl. obtained from media with different concentrations of lanthanum nitrate respectively.

[0179] Experimental Example 6

[0180] This experimental example involves an experiment on the effect of lanthanum nitrate on the stem cells of Dendrobium nobile Lindl.

[0181] Set Comparative Example 9: Do not add lanthanum nitrate to the B5 medium, and the others are the same as in Example 13.

[0182] Comparative Example 10: Do not add lanthanum nitrate to the MS medium, and the others are the same as in Example 14.

[0183] Weigh the finally obtained stem cells of Dendrobium nobile Lindl. in the above Examples 13 and 14 and Comparative Examples 9 and 10 respectively. Conduct 5 experiments for each group and finally take the average value. The results are shown in Table 7.

[0184] Table 7 Comparison results of the weights of stem cells of Dendrobium nobile Lindl.

[0185] Example 13 Comparative Example 9 Example 14 Example 14 Example 14 Comparative Example 10 Lanthanum nitrate concentration / mg / L 300 0 100 200 300 0 Culture medium type B5 B5 MS MS MS MS Weight / g 88.13 77.23 98.56 104.44 117.13 89.43

[0186] As can be seen from Table 7, the weight ratio of the Dendrobium officinale stem cells obtained in Examples 13 and 14 is greater than that in Proportions 9 and 10, indicating that the culture medium supplemented with lanthanum nitrate at a concentration of 100 - 300 mg / L can cultivate a large number of stem cells in a relatively short time. The weight of the stem cells obtained from the culture medium with different concentrations of lanthanum nitrate in Example 14 is significantly better than that in Example 13, indicating that the composition and ratio of the culture medium in Example 14 are more suitable for cultivating a large number of Dendrobium officinale stem cells in a short time.

[0187] Example 15

[0188] This example takes Dendrobium officinale as an example and relates to the method for culturing and collecting Dendrobium officinale stem cells under the condition of B5 culture medium supplemented with lanthanum nitrate. The specific steps are as follows:

[0189] (1) Pretreatment of young stems: First, take the young stems of Dendrobium officinale and rinse them with clean water for 3 h, and carefully brush the parts that are not easy to clean with a brush to remove dust and other attachments. Then, wash and disinfect them with an aqueous alcohol solution with a volume concentration of 75% for 20 s, and then rinse them 3 times with sterile water. Next, disinfect them with a disinfectant containing sodium hypochlorite for 5 min, and finally rinse them 3 times with sterile water for standby;

[0190] (2) Obtaining of stem cells: Cut off the stem tips of the disinfected young stems in step (1) with a surgical blade in a biosafety cabinet, layer by layer peel the stem tips with a dissecting needle and forceps, leaving 5 - 6 leaf primordia, and then cut at 0.5 mm from the stem tip with a surgical blade. In a sufficient interval space, the stem tips with the meristem of Dendrobium officinale can be picked out with a dissecting needle, and then the separated stem tips are put into a triangular flask containing the culture medium for cultivation. The culture medium is B5 medium containing 0.100 mg / L of 6 - BA, 15 mg / L of sucrose, 0.50 mg / L of methyl jasmonate and 0.100 mg / L of triacontanol. In addition, the culture medium is supplemented with lanthanum nitrate with a final concentration of 300 mg / L, the pH value of the culture medium is 6.2, the triangular flask is placed on a vertical constant temperature oscillator, the rotation speed is 110 r / min, and the temperature is 22 °C for shaking culture for 3 days; after the culture is completed, first filter out the larger flakes and fragments with a No. 2 sieve, and then perform secondary filtration separation with a 40 - um cell filter membrane. The filtrate is the filtrate containing stem cells;

[0191] (3) Expansion culture of stem cells: Transfer the finally filtered filtrate containing stem cells into an Erlenmeyer flask, and add B5 medium containing 0.100 mg / L of 6-BA, 15 mg / L of sucrose, 0.50 mg / L of methyl jasmonate, and 0.100 mg / L of triacontanol. Additionally, add lanthanum nitrate with a concentration of 300 mg / L to the medium, and place it in a vertical constant temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C for shaking culture for 2 days. After the culture is completed, transfer the medium containing stem cells evenly from 1 Erlenmeyer flask to 3 Erlenmeyer flasks, and continue to add B5 medium containing 0.100 mg / L of 6-BA, 15 mg / L of sucrose, 0.50 mg / L of methyl jasmonate, and 0.100 mg / L of triacontanol (lanthanum nitrate with a concentration of 300 mg / L is also added to the medium) until the amount of medium in each Erlenmeyer flask reaches about 1 / 3 of the position, and perform subculture in a vertical constant temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C;

[0192] (4) Collection of stem cells: After 48 h of subculture of stem cells, collect them. Pour the medium containing stem cells into a centrifuge bottle, balance it, place it in a centrifuge with a rotation speed of 2200 r / min and a centrifugation time of 10 minutes. After centrifugation, discard the supernatant to obtain the stem cells of Dendrobium officinale. Freeze-dry or microwave-dry the obtained stem cells. After complete drying, use a small pulverizer to pulverize and sieve to obtain the Dendrobium officinale stem cell powder.

[0193] Example 16

[0194] This example takes Dendrobium officinale as an example and relates to a method for culturing and collecting Dendrobium officinale stem cells under the condition of MS medium added with lanthanum nitrate. The specific steps are as follows:

[0195] (1) Pretreatment of young stems: First, take the young stems of Dendrobium officinale and rinse them with clean water for 3 h, and carefully brush the parts that are not easy to clean with a brush to remove dust and other attachments. Then, wash and disinfect them with an aqueous alcohol solution with a volume concentration of 75% for 20 s, and then rinse them with sterile water 3 times. Next, disinfect them with a disinfectant containing sodium hypochlorite for 5 min, and finally rinse them with sterile water 3 times for standby;

[0196] (2) Obtaining stem cells: After disinfecting the tender stems in step (1), cut off the shoot tips with a surgical blade in a biosafety cabinet. Then, layer by layer peel the shoot tips with a dissecting needle and forceps, leaving 5 - 6 leaf primordia. Next, cut at 0.5 mm from the shoot tip with a surgical blade. In a sufficient interval space, pick out the shoot tips with the Dendrobium officinale Kimura et Migo meristem with a dissecting needle. Then, transfer the separated shoot tips into 3 Erlenmeyer flasks containing culture medium. The culture medium is MS medium containing amphotericin B at a concentration of 1.0 mg / L, double antibody (100X penicillin - streptomycin mixture) at a volume concentration of 1.0%, 1.0 mg / L of 6 - BA, and 1.0 mg / L of NAA. Lanthanum nitrate at concentrations of 100 mg / L, 200 mg / L, and 300 mg / L is respectively added to the 3 culture media. Place the Erlenmeyer flasks in a vertical constant - temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C for shaking culture for 3 days. After the culture, first filter out the larger flakes and debris with a No. 2 sieve, and then perform secondary filtration separation with a 40 - μm cell filter membrane. The filtrate is the filtrate of stem cells;

[0197] (3) Sub - culturing of stem cells: Transfer the finally filtered filtrate containing stem cells into an Erlenmeyer flask, and add MS medium containing amphotericin B at a concentration of 1.0 mg / L, double antibody at a volume concentration of 1.0%, 1.0 mg / L of 6 - BA, and 1.0 mg / L of NAA. Lanthanum nitrate at concentrations of 100 mg / L, 200 mg / L, and 300 mg / L is respectively added to the 3 culture media. Place the Erlenmeyer flask in a vertical constant - temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C for shaking culture for 2 days. After the culture, evenly transfer the culture medium containing stem cells from 1 Erlenmeyer flask to 3 Erlenmeyer flasks, and continue to add MS medium containing amphotericin B at a concentration of 1.0 mg / L, double antibody at a volume concentration of 1.0%, 1.0 mg / L of 6 - BA, and 1.0 mg / L of NAA (lanthanum nitrate at concentrations of 100 mg / L, 200 mg / L, and 300 mg / L is respectively added to the other 3 culture media) until the amount of the culture medium in each Erlenmeyer flask reaches about 1 / 3 of the position. Then, perform sub - culturing in a vertical constant - temperature oscillator with a rotation speed of 110 r / min and a temperature of 22 °C;

[0198] (4) Collection of stem cells: The stem cells were subcultured for 48 h and then collected. The culture medium containing the stem cells was poured into a centrifuge bottle. After balancing, it was placed in a centrifuge at a rotation speed of 2200 r / min for 10 minutes. After centrifugation, the supernatant was discarded, and the stem cells of Dendrobium officinale were obtained. The obtained stem cells were freeze-dried or microwave-dried. After complete drying, they were pulverized with a small pulverizer and sieved to obtain the stem cell powder of Dendrobium officinale. The stem cell powders of Dendrobium officinale obtained from culture media with different concentrations of lanthanum nitrate were collected respectively.

[0199] Experimental Example 7

[0200] This experimental example relates to an experiment on the effect of lanthanum nitrate on the stem cells of Dendrobium officinale.

[0201] Set Control Example 11: Lanthanum nitrate was not added to the B5 medium, and the others were the same as in Example 15.

[0202] Control Example 12: Lanthanum nitrate was not added to the MS medium, and the others were the same as in Example 16.

[0203] The finally obtained stem cells of Dendrobium officinale in the above Examples 15 and 16 and Control Examples 11 and 12 were weighed separately. Each group was experimented 5 times, and the average value was finally taken. The results are shown in Table 8.

[0204] Table 8 Comparison results of the weights of the stem cells of Dendrobium officinale

[0205] Example 15 Comparative Example 11 Example 16 Example 16 Example 16 Comparative Example 12 Lanthanum nitrate concentration / mg / L 300 0 100 200 300 0 Culture medium type B5 B5 MS MS MS MS Weight / g 82.22 76.23 94.56 103.44 116.13 84.43

[0206] It can be seen from Table 8 that the weights of the stem cells of Dendrobium officinale obtained in Examples 15 and 16 are greater than those in Control Examples 11 and 12, indicating that the culture medium added with lanthanum nitrate at a concentration of 100 - 300 mg / L can culture a large number of stem cells in a shorter time. The weights of the stem cells obtained from the culture media with different concentrations of lanthanum nitrate in Example 16 are significantly better than those in Example 15, indicating that the composition and ratio of the culture medium in Example 16 are more suitable for culturing a large number of stem cells of Dendrobium officinale in a short time.

[0207] In view of the fact that the current Orchidaceae plants are difficult to reproduce due to environmental and other factors, it is urgent to develop a method that can stably obtain plant stem cells with various active ingredients. The culture medium added with lanthanum nitrate at a concentration of 100 - 300 mg / L in this invention can culture a large number of stem cells in a shorter time. It can be found by microscopic examination of staining that the degree of cell dispersion and the number are better than others, providing a basis for the next subculture and proliferation culture.

[0208] The above are only the preferred embodiments of the present invention, and there is no limitation to the present invention in any form. Although the present invention has been disclosed above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art of the present invention can make some changes or modifications to equivalent embodiments with equivalent changes by using the technical content prompted above within the scope of the technical solution of the present invention. However, as long as it does not depart from the content of the technical solution of the present invention, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention still fall within the scope of the present invention's solution.

Claims

1. A culture medium for orchid plant stem cells, characterized in that, The culture medium includes a basal medium, and lanthanum nitrate at 100 - 300 mg / L is added to the basal medium; the basal medium is selected from one of MS medium and B5 medium.

2. The culture medium according to claim 1, wherein, The basal medium is MS medium, and amphotericin B at 0.9 - 1.2 mg / L, double antibody with a volume concentration of 0.8% - 1.3% (the double antibody is 100X penicillin - streptomycin mixture), 6 - BA at 0.9 - 1.1 mg / L, and NAA at 0.9 - 1.2 mg / L are also added thereto.

3. The culture medium according to claim 2, characterized in that, 1.0 mg / L of amphotericin B, double antibody with a volume concentration of 1.0%, 1.0 mg / L of 6 - BA, and 1.0 mg / L of NAA are added to the MS medium.

4. The culture medium according to claim 1, characterized in that, The basal medium is B5 medium, and 6 - BA at 0.099 - 0.130 mg / L, sucrose at 12 - 17 mg / L, methyl jasmonate at 0.49 - 0.54 mg / L, and triacontanol at 0.098 - 0.120 mg / L are also added thereto.

5. The culture medium according to claim 4, characterized in that, 0.100 mg / L of 6 - BA, 15 mg / L of sucrose, 0.50 mg / L of methyl jasmonate, and 0.100 mg / L of triacontanol are added to the B5 medium.

6. A method for culturing orchid plant stem cells, characterized in that, It includes: (1) Pretreatment of explants: Take the tender stems of orchid plants, wash and disinfect them for later use; (2) Obtaining of stem cells: Dissect the meristem from the shoot tip after being treated in step (1), transfer it to a culture medium for culturing. The culture medium includes a basal medium, and lanthanum nitrate at 100 - 300 mg / L is added to the basal medium; the basal medium is selected from one of MS medium and B5 medium; after the culture is completed, filter to obtain a filtrate containing stem cells; (3) Sub - culture: Transfer the filtrate containing stem cells obtained in step (2) to a culture medium for culturing. After the culture is completed, divide the culture medium containing stem cells into several portions, transfer them to several culture containers, and then add culture media respectively for sub - culturing; (4) Collection: Centrifuge the stem cell culture solution obtained in step (3), discard the supernatant, and thus obtain the stem cells of orchid plants.

7. The cultivation method according to claim 6, characterized in that, In steps (2) and (3), the culture medium includes: The basal medium is MS medium, and lanthanum nitrate at 100 - 300 mg / L, amphotericin B at 0.9 - 1.2 mg / L, double antibody with a volume concentration of 0.8% - 1.3% (the double antibody is 100X penicillin - streptomycin mixture), 6 - BA at 0.9 - 1.1 mg / L, and NAA at 0.9 - 1.2 mg / L are added thereto; or, the culture medium includes: The basal medium is B5 medium, and lanthanum nitrate at 100 - 300 mg / L, 6 - BA at 0.099 - 0.130 mg / L, sucrose at 12 - 17 mg / L, methyl jasmonate at 0.49 - 0.54 mg / L, and triacontanol at 0.098 - 0.120 mg / L are added thereto.

8. The cultivation method according to claim 6 or 7, characterized in that, In step (1), the washing and disinfection include: Rinse the shoot tips with clear water, then wash them with an aqueous alcohol solution with a volume concentration of 75%, wash with sterile water, then disinfect with a disinfectant containing sodium hypochlorite, and then wash with sterile water.

9. The culturing method according to claim 6 or 7, characterized in that, In steps (2) and (3), the temperature for culturing is 20 - 25 °C, the shaker speed is 80 - 200 r / min, and the culturing time is 3 - 7 days.

10. The culture medium according to any one of claims 1-5 or the culture method according to claim 6 or 7, characterized in that, The said Orchidaceae plants include ornamental orchids, Dendrobium officinale, Dendrobium loddigesii, Dendrobium fimbriatum, Dendrobium chrysanthum, Dendrobium nobile, Dendrobium huoshanense, Dendrobium moniliforme, Dendrobium strongylanthum, Dendrobium wardianum, Dendrobium aduncum, Dendrobium denneanum.