Method for cultivating dendrobium officinale tender shoots under weak sugar and ventilation conditions

Abstract

The invention discloses a method for cultivating dendrobium officinale tender shoots under weak sugar and ventilation conditions, and belongs to the technical field of dendrobium planting. The formula of the dendrobium officinale tender shoot induction and proliferation liquid culture medium is optimized, and a certain amount of natural extracts and growth hormones are added into the improved MS culture medium to improve the growth promoting effect of the dendrobium officinale tender shoots; in the suspension culture process, a certain amount of air and carbon dioxide gas are additionally introduced into a culture medium, so that the nutrient absorption rate of a culture can be improved, the photosynthesis efficiency of protocorm is promoted, the growth speed of dendrobium officinale tender shoots is increased, the biomass accumulation is enhanced, the tender shoot induction rate and the proliferation coefficient of tissue culture are further improved, and the survival rate of the dendrobium officinale tender shoots is increased. Stable characters of the tender shoots are kept, and the content of active ingredients such as polysaccharide and amino acid in the tender shoots is improved; in addition, by means of ventilation co-culture, the using amount of sugar in the culture medium can be reduced, the culture cost and the pollution probability are reduced, the time needed by the dendrobium officinale tender shoot multiplication culture stage can be effectively shortened, and the culture cost is reduced. In addition, the dendrobium officinale tender shoots cultivated by the method also show a good analgesic effect, and have a very good market application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of Dendrobium cultivation, and particularly relates to a method for cultivating tender buds of Dendrobium officinale under conditions of weak sugar and ventilation. Moreover, the application of the tender buds of Dendrobium officinale cultivated by this method in analgesia is disclosed. Background Art

[0002] Dendrobium officinale is a perennial herbaceous epiphyte of the genus Dendrobium in Orchidaceae. It contains various amino acids, rich trace elements, Dendrobium polysaccharides, bibenzyl compounds and other medicinal components, and has the effects of inhibiting tumors, enhancing human immunity, and reducing blood sugar. It is a traditional precious Chinese medicinal material in China.

[0003] With the improvement of people's awareness of health care and cultivation, the market demand for Dendrobium officinale has also increased significantly. However, the seeds of Dendrobium officinale are extremely small and without endosperm, and the germination rate under natural conditions is extremely low, making it difficult to cultivate with seedlings. Although traditional methods such as ramet division and cutting have relatively low costs, their propagation rates are also low, and the growth cycle is long, which is obviously extremely disadvantageous for the large-scale production of Dendrobium officinale in the market. In order to meet the growing demand for Dendrobium officinale, it is extremely necessary to achieve the rapid propagation and large-scale production of Dendrobium officinale.

[0004] Tissue culture technology is an asexual propagation technology that inoculates in vitro plant tissues (such as shoot tips, leaves, endosperm, etc.) into artificial media through aseptic operation, induces their dedifferentiation to form callus, and then further differentiates into complete plants. This technology has been successfully applied to the large-scale propagation of Dendrobium officinale.

[0005] There are two main categories of commonly used tissue culture methods, namely cultivating tissue culture seedlings through protocorms and increasing the yield of tissue culture seedlings through the proliferation of clustered buds. Cultivating tissue culture seedlings through protocorms mainly includes steps such as the selection (shoot tips, lateral buds or seeds of healthy plants) and disinfection of explants, protocorm induction, protocorm proliferation, differentiation culture (including bud differentiation and root induction), acclimatization and transplantation, etc. To improve the germination rate of Dendrobium officinale explants, as well as the induction rate and proliferation coefficient of tender buds, it is necessary to select, prepare and use a suitable culture medium during tissue culture, and certain improvements need to be made to tissue culture technology to optimize the process of cultivating tissue culture seedlings, establish a feasible proliferation system for tender buds of Dendrobium officinale, so as to achieve the ultimate goal of increasing the yield of Dendrobium officinale. If the growth and cultivation cycle can be shortened synchronously during this period, it will provide more valuable technical information and scientific basis for factory-scale rapid seedling raising.

[0006] In the prior art, the focus on how to improve the cultivation effect of Dendrobium officinale mainly lies in the improvement of the culture medium. The common method is to add a certain amount of growth hormone to the basic MS or 1 / 2MS culture medium to enhance the cultivation effect. For example, a tissue culture and rapid propagation method of Dendrobium officinale disclosed in Chinese Patent CN 116965335 B uses stem segments as explants. The optimal culture medium for protocorm formation is prepared by adding 6-benzylaminopurine (6-BA), naphthaleneacetic acid (NAA), kinetin (KT), sucrose and agar to the basic MS culture medium; the proliferation and differentiation culture medium is prepared by adding 6-BA and NAA to the basic MS culture medium; through the improvement of the culture medium, the induction rate, differentiation rate and rooting rate of Dendrobium officinale protocorms all reach more than 85%. However, the obvious deficiency of cultivating using this scheme is that it takes more than 60 days from the formation of protocorms to the growth of sprouted buds, and the cycle is relatively long, which is not conducive to increasing the yield of Dendrobium in the marketization stage and cannot well meet the market demand.

[0007] Therefore, when improving the culture medium formula, in addition to paying attention to data such as the induction rate and proliferation coefficient, the cultivation cycle should also be focused on. The cultivation cycle should be shortened as much as possible to better meet the marketization demand. However, to achieve this, in addition to improving the culture medium formula, it may also be necessary to conduct in-depth exploration and research on the cultivation method. Summary of the Invention

[0008] In view of the above problems, the present invention aims to provide a method for cultivating Dendrobium officinale tender buds under weak sugar and aeration conditions, which not only adjusts the culture medium formula used in the cultivation process of the induction and proliferation stages of Dendrobium officinale tender buds, but also improves the cultivation method. Specifically, a certain amount of air and carbon dioxide are continuously introduced into the culture medium during the tissue culture process for auxiliary cultivation. Under the dual improvements, the induction rate and proliferation rate of Dendrobium officinale tender buds can be effectively improved, the traits of Dendrobium officinale tender buds can be kept stable, the cultivation cycle can be shortened, the sugar consumption in the culture medium can be reduced, the cultivation cost can be effectively reduced, and it has good application prospects.

[0009] To achieve the above object, the technical solution adopted by the present invention is as follows: A method for cultivating Dendrobium officinale tender buds under weak sugar and aeration conditions, comprising the following steps:

[0010] 1) Inoculate the disinfected Dendrobium officinale seeds into the protocorm induction culture medium to obtain Dendrobium officinale protocorms;

[0011] 2) Inoculate the Dendrobium officinale protocorms into the tender bud induction liquid culture medium, and continuously introduce air and carbon dioxide gas into the culture medium during the induction culture process to obtain induced small corms;

[0012] 3) Inoculate the cormels obtained in the previous step into the liquid medium for shoot proliferation. During the proliferation culture process, continuously introduce air and carbon dioxide gas into the medium to obtain Dendrobium officinale shoots.

[0013] Furthermore, in step 2), the inoculation amount of protocorms is 12 - 14 g / L, the temperature during the shoot induction culture process is 21 - 26 °C, the relative air humidity is 45 - 55%, and the light duration is 12 - 14 h / day.

[0014] Furthermore, in step 2), the compressed air introduced into the medium during the induction culture process has an aeration rate of 3 - 4 L / min and a flow rate of 10 - 12 m / s; the concentration of carbon dioxide in the culture vessel is adjusted according to the light and dark cycles. The concentration of carbon dioxide in the vessel during the light period is 600 - 800 ppm, and it is adjusted to 300 - 500 ppm during the dark period.

[0015] Furthermore, in step 3), the inoculation amount of cormels is 10 - 12 g / L, the temperature during the shoot proliferation culture process is 20 - 26 °C, the relative air humidity is 55 - 60%, and the light duration is 14 - 16 h / day.

[0016] Furthermore, in step 3), the compressed air introduced into the medium has an aeration rate of 4 - 5 L / min and a flow rate of 10 - 12 m / s; the concentration of carbon dioxide in the culture vessel is adjusted according to the light and dark cycles. The concentration of carbon dioxide in the vessel during the light period is 600 - 800 ppm, and it is adjusted to 300 - 500 ppm during the dark period.

[0017] Furthermore, in step 2), the preparation method of the liquid medium for shoot induction is as follows: Measure 150 - 350 mL of potato extract, supplement distilled water to 500 - 700 mL, and sequentially add 30 - 40 mL of coconut water, 1.5 - 2.5 mL of the first organic element stock solution, 5 - 15 mL of the sulfur, nitrogen, and phosphorus element stock solution, 5 - 15 mL of the calcium salt stock solution, 5 - 15 mL of the ammonium salt stock solution, 0.5 - 1.5 mL of the trace element stock solution, 5 - 15 mL of the first iron salt stock solution, 0.2 - 1.5 mL of the 6 - BA stock solution, and 0.2 - 0.6 mL of the NAA stock solution. Add 15 - 20 g of sucrose, stir and mix evenly, make up the volume to 1000 mL with distilled water, adjust the pH of the solution to 6.10, and sterilize at 121 °C for 21 minutes.

[0018] Further, in step 3), the method for preparing the tender shoot proliferation liquid medium is as follows: Measure 150 - 350 mL of potato extract, supplement distilled water to 500 - 700 mL, and sequentially add 30 - 40 mL of coconut water, 1.5 - 2.5 mL of the first organic element mother liquor, 5 - 15 mL of the sulfur - nitrogen - phosphorus element mother liquor, 5 - 15 mL of the calcium salt mother liquor, 5 - 15 mL of the ammonium salt mother liquor, 0.5 - 1.5 mL of the trace element mother liquor, 5 - 15 mL of the first iron salt mother liquor, 0.15 - 0.45 mL of the 6 - BA mother liquor, 5 - 10 g of sucrose and 3 - 5 g of edible glucose, stir and mix evenly, make up the volume to 1000 mL with distilled water, adjust the pH of the solution to 6.10, and sterilize at 121 °C for 21 minutes.

[0019] Further, the preparation methods of different element mother liquors and potato extract are as follows:

[0020] Each liter of the first organic element mother liquor contains: 1 g of glycine, 0.25 g of pyridoxine hydrochloride, 0.05 g of thiamine hydrochloride, 0.25 g of nicotinic acid, 50 g of inositol, and the balance is deionized water;

[0021] Each liter of the sulfur - nitrogen - phosphorus element mother liquor contains: 37 g of magnesium sulfate, 190 g of potassium nitrate, 17 g of potassium dihydrogen phosphate, and the balance is deionized water;

[0022] Each liter of the calcium salt mother liquor contains: 44 g of calcium chloride, 28 g of calcium nitrate, and the balance is deionized water;

[0023] Each liter of the ammonium salt mother liquor contains: 48.5 g of ammonium sulfate, and the balance is deionized water;

[0024] Each liter of the first iron salt mother liquor contains: 3.45 g of ferrous sulfate, 3.73 g of disodium ethylenediaminetetraacetate, and the balance is deionized water;

[0025] Each liter of the trace element mother liquor contains: 16.9 g of manganese sulfate, 8.6 g of zinc sulfate, 6.2 g of boric acid, 0.83 g of potassium iodide, 0.25 g of sodium molybdate, 0.025 g of copper sulfate, 0.025 g of cobalt chloride, and the balance is deionized water;

[0026] 6 - BA mother liquor: 80 - 120 mg of 6 - BA is dissolved in 5 - 15 mL of an HCl solution with a concentration of 0.5 - 1.5 mol / L, and the volume is made up to 50 - 150 mL with deionized water;

[0027] NAA mother liquor: 30 - 60 mg of naphthylacetic acid is dissolved in 15 - 25 mL of an ethanol solution, and the volume is made up to 50 - 110 mL with deionized water;

[0028] Potato extract: Wash and peel the potatoes and cut them into slices. Put 150 - 200 g of sliced potatoes into 350 - 450 mL of distilled water and steam for 20 - 30 min, then filter and collect the filtrate.

[0029] Preferably, the preparation method of the tender bud induction liquid medium is as follows: Measure 350 mL of potato extract, supplement distilled water to 600 mL, and sequentially add 35 mL of coconut water, 2 mL of the first organic element mother liquor, 10 mL of the sulfur-nitrogen-phosphorus element mother liquor, 10 mL of the calcium salt mother liquor, 10 mL of the ammonium salt mother liquor, 1 mL of the trace element mother liquor, 10 mL of the first iron salt mother liquor, 1 mL of the 6-BA mother liquor, and 0.4 mL of the NAA mother liquor. Add 18 g of sucrose, stir and mix well, make up the volume to 1000 mL with distilled water, adjust the pH of the solution and then sterilize it;

[0030] The preparation method of the tender bud proliferation liquid medium is as follows: Measure 350 mL of potato extract, supplement distilled water to 600 mL, and sequentially add 35 mL of coconut water, 2 mL of the first organic element mother liquor, 10 mL of the sulfur-nitrogen-phosphorus element mother liquor, 10 mL of the calcium salt mother liquor, 10 mL of the ammonium salt mother liquor, 1 mL of the trace element mother liquor, 10 mL of the first iron salt mother liquor, 0.3 mL of the 6-BA mother liquor, 7 g of sucrose, and 5 g of edible glucose. Stir and mix well, make up the volume to 1000 mL with distilled water, adjust the pH of the solution and then sterilize it.

[0031] The beneficial effects of the present invention are as follows:

[0032] 1. The present application adjusts the formulas of the tender bud induction liquid medium and the tender bud proliferation liquid medium. Compared with the basic MS medium, not only the dosages of elements are adjusted, but also a certain amount of natural extracts (potato extract, coconut water) and growth hormones (6-BA, NAA) are additionally added to the medium. The growth promotion effect is obvious. It not only improves the induction rate and proliferation coefficient of Dendrobium officinale tender buds, but also can keep the traits of Dendrobium officinale tender buds stable, so that the content of active ingredients in Dendrobium officinale tender buds is significantly increased;

[0033] 2. In addition to adjusting the medium formula, the present application also improves the culture method. During the suspension culture of Dendrobium officinale tender buds, a certain amount of air and carbon dioxide gas are additionally introduced into the medium for auxiliary combined culture. This method can help improve the nutrient absorption rate of protocorms, promote the photosynthesis efficiency of protocorms and optimize the metabolic environment, thereby accelerating the growth rate of Dendrobium officinale tender buds and enhancing the biomass accumulation. Compared with the scheme of non-additional aeration for combined culture, this auxiliary culture scheme helps to further improve the induction rate and proliferation coefficient of Dendrobium officinale tender buds by tissue culture, and can further promote the increase of the content of active ingredients such as polysaccharides and amino acids in Dendrobium officinale tender buds;

[0034] 3. This application proposes a cultivation plan for aerated hypoglycemic Dendrobium officinale. During the cultivation of Dendrobium officinale tender buds based on an improved hypoglycemic culture medium, a certain amount of CO2 gas is additionally introduced into the culture vessel as a supplementary carbon source, innovating the carbon source supply mode. Different from the traditional cultivation method that relies on sugar as the main carbon source, this method can achieve the goal of improving the quality and yield of Dendrobium officinale tender stems through the combined cultivation method of passing CO2 in a low-sugar environment and the precise control of the development environmental conditions. Moreover, the reduction in sugar usage can effectively reduce the raw material cost and the probability of microbial contamination of the culture medium;

[0035] 4. This application conducts tissue culture of Dendrobium officinale tender buds based on the improved MS medium, and continuously introduces air and carbon dioxide gas into the medium during the tender bud induction and proliferation culture processes for auxiliary culture. Under the combined action, it can effectively shorten the time required for the proliferation culture stage of Dendrobium officinale tender buds, help shorten the entire cultivation cycle, significantly increase the yield of Dendrobium in the marketization stage, reduce the cultivation cost, and quickly respond to market demands, having very good market application prospects;

[0036] 5. The Dendrobium officinale tender buds cultivated by the method disclosed in this application have stable traits and high functional component contents. After making them into dry powder products and applying them to organisms, it is found that they have good analgesic treatment effects and have good medical prospects. Description of the Drawings

[0037] Figure 1 It is a photo of the Dendrobium officinale tender buds cultivated in Example 1;

[0038] Figure 2 It is a schematic structural diagram of a cultivation barrel, where 1 - cultivation barrel, 2 - air inlet pipe, 3 - carbon dioxide inlet pipe, 4 - exhaust pipe, 5 - feed pipe;

[0039] Figure 3 It is Figure 2 The enlarged view of part A in

[0040] Figure 4 It is a comparison chart of the writhing times (A) and writhing latency (B) of each group of mice 20 minutes after injecting acetic acid. *** indicates a significant difference from the control group with P < 0.001, ** indicates a significant difference from the control group with P < 0.01, * indicates a significant difference from the control group with P < 0.05, and ns indicates a significant difference from the control group with P ≥ 0.05. Detailed Embodiments

[0041] In order to enable ordinary technicians in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be further described below in conjunction with the drawings and embodiments.

[0042] Example 1

[0043] This embodiment discloses a tissue culture method for the tender buds of Dendrobium officinale, which specifically includes several culture stages: protocorm induction - tender bud induction - tender bud proliferation. Different culture stages will apply protocorm induction medium, tender bud induction liquid medium and tender bud proliferation liquid medium.

[0044] Among them, the following elemental mother liquors are required for preparing the tender bud induction liquid medium and the tender bud proliferation liquid medium:

[0045] First organic elemental mother liquor ( / L): glycine 1g, pyridoxine hydrochloride 0.25g, thiamine hydrochloride 0.05g, nicotinic acid 0.25g, inositol 50g, with the balance being deionized water;

[0046] Sulfur, nitrogen, and phosphorus elemental mother liquor ( / L): magnesium sulfate (MgSO4·7H2O) 37g, potassium nitrate 190g, potassium dihydrogen phosphate 17g, with the balance being deionized water;

[0047] Calcium salt mother liquor ( / L): calcium chloride (CaCl2·2H2O) 44g, calcium nitrate 28g, with the balance being deionized water;

[0048] Ammonium salt mother liquor ( / L): ammonium sulfate 48.5g, with the balance being deionized water;

[0049] First iron salt mother liquor ( / L): ferrous sulfate (FeSO4·7H2O) 3.45g, disodium ethylenediaminetetraacetate (Na2-EDTA) 3.73g, with the balance being deionized water;

[0050] Trace element mother liquor ( / L): manganese sulfate (MnSO4·4H2O) 16.9g, zinc sulfate (ZnSO4·7H2O) 8.6g, boric acid 6.2g, potassium iodide 0.83g, sodium molybdate 0.25g, copper sulfate 0.025g, cobalt chloride 0.025g, with the balance being deionized water;

[0051] 6-Benzylaminopurine (6-BA) mother liquor: 100 mg of 6-BA is dissolved in 10 mL of HCl solution (1 mol / L), and the volume is made up to 100 mL with deionized water;

[0052] Naphthaleneacetic acid (NAA) mother liquor: 50 mg of naphthaleneacetic acid is dissolved in 20 mL of ethanol solution (95%), and the volume is made up to 100 mL with deionized water;

[0053] Potato extract: Wash and peel the potatoes and cut them into slices. Put 200 g of potato slices into 400 mL of distilled water and steam for 25 minutes, and collect the filtrate after filtration.

[0054] The formula of the tender bud induction liquid medium is as follows:

[0055] Measure 350 mL of potato extract, supplement distilled water to 600 mL, and sequentially add 35 mL of coconut water, 2 mL of the first organic element mother liquor, 10 mL of the sulfur-nitrogen-phosphorus element mother liquor, 10 mL of the calcium salt mother liquor, 10 mL of the ammonium salt mother liquor, 1 mL of the trace element mother liquor, 10 mL of the first iron salt mother liquor, 1 mL of the 6-BA mother liquor, and 0.4 mL of the NAA mother liquor. Add 18 g of sucrose, stir and mix well, make up the volume to 1000 mL with distilled water, adjust the pH of the solution to 6.10, and sterilize at 121 °C and 1.1 MPa for 21 minutes to obtain the shoot induction liquid medium. After sterilization, the pH of the medium will decrease to 5.6 - 5.8.

[0056] The formula for the shoot proliferation liquid medium is as follows:

[0057] Measure 350 mL of potato extract, supplement distilled water to 600 mL, and sequentially add 35 mL of coconut water, 2 mL of the first organic element mother liquor, 10 mL of the sulfur-nitrogen-phosphorus element mother liquor, 10 mL of the calcium salt mother liquor, 10 mL of the ammonium salt mother liquor, 1 mL of the trace element mother liquor, 10 mL of the first iron salt mother liquor, 0.3 mL of the 6-BA mother liquor, 7 g of sucrose, and 5 g of edible glucose. Stir and mix well, make up the volume to 1000 mL with distilled water, adjust the pH of the solution to 6.10, and sterilize at 121 °C and 1.1 MPa for 21 minutes to obtain the shoot proliferation liquid medium.

[0058] The following mother liquors are required for preparing the protocorm induction medium:

[0059] The second organic element mother liquor ( / L): glycine 0.2 g, pyridoxine hydrochloride 0.05 g, thiamine hydrochloride 0.01 g, nicotinic acid 0.05 g, inositol 10 g, and the balance is deionized water;

[0060] The macroelement mother liquor ( / L): potassium nitrate 38 g, ammonium sulfate 16.5 g, magnesium sulfate (MgSO4·7H2O) 7.4 g, potassium dihydrogen phosphate 3.4 g, calcium chloride (CaCl2·2H2O) 8.8 g, and the balance is deionized water;

[0061] The trace element mother liquor: the same as the trace element mother liquor used in preparing the shoot induction liquid medium;

[0062] The second iron salt mother liquor ( / L): ferrous sulfate (FeSO4·7H2O) 5.56 g, disodium ethylenediaminetetraacetate (Na2-EDTA) 7.46 g, and the balance is deionized water;

[0063] The naphthylacetic acid (NAA) mother liquor: 50 mg of naphthylacetic acid is dissolved in 20 mL of ethanol solution (95%), and the volume is made up to 100 mL with deionized water.

[0064] The formula for the protocorm induction medium is as follows:

[0065] Add 75 g of water to 30 g of potatoes after cleaning and peeling, and process them with a juicer to obtain mashed potatoes. Pour the mashed potatoes into a preparation container, and sequentially add 25 mL of macroelement mother liquor, 1 mL of microelement mother liquor, 10 mL of second organic element mother liquor, 5 mL of second iron salt mother liquor, 0.2 mL of naphthaleneacetic acid mother liquor, 30 g of sucrose, and 5 g of agar powder. Stir well and make up the volume to 1000 mL with distilled water. Adjust the pH to 5.9 and sterilize at 121 °C for 21 min to obtain the protocorm induction medium.

[0066] The specific process of cultivating Dendrobium officinale buds is as follows:

[0067] S1. Select mature and unopened Dendrobium officinale seeds, inoculate them into the protocorm induction medium after disinfection treatment, and culture them at a temperature of 20-26 °C, with a relative air humidity of 55-65%, a light duration of 10 h / day, and a light intensity maintained at 1500 Lux to obtain Dendrobium officinale protocorms; it is found through testing that the temperature and humidity conditions will fluctuate to a certain extent with the adjustment of the light period and dark period, but as long as the fluctuations are within the above range, the impact on the final culture effect is not significant.

[0068] S2. Inoculate the Dendrobium officinale protocorms obtained in the previous step into the bud induction liquid medium at an inoculation density of 12 g / L. The medium is placed in a relatively sealed culture barrel 1 (the structure of the culture barrel refers to Figure 2 、 Figure 3)So that the induction process of Dendrobium officinale young shoots can be completed in a relatively enclosed environment to reduce the influence of the external environment on the culture process. The filling volume of the culture medium in the barrel is 8.5 L. When the amount of the culture medium is less than 6 L, liquid supplementation is required, and the liquid supplementation volume is 4 L (determined by the liquid level scale on the barrel body). The culture temperature is 21 - 26 °C, the relative air humidity is 45 - 55%, the light time is 12 h / day, and the light intensity is 2600 Lux (the temperature and humidity fluctuations within the corresponding ranges have little impact. In principle, the temperature is slightly higher during the light period and slightly lower during the dark period); An air inlet pipe 2, a carbon dioxide inlet pipe 3, an exhaust pipe 4, and a feed pipe 5 that penetrate inside and outside the barrel body are connected to the culture barrel 1. One-way valves are provided on different pipes. The air inlet pipe 2 and the carbon dioxide inlet pipe 3 are inserted below the liquid level. The feed pipe 5 is used to input protocorms into the barrel. After the explants are introduced, the one-way valve on the feed pipe 5 is closed; During the induction culture process of Dendrobium officinale young shoots, filtered compressed air is injected into the liquid culture medium in the culture barrel 1 through the air inlet pipe 2, with an aeration volume of 3 L / min and a flow rate of 12 m / s; While injecting air, carbon dioxide gas is additionally injected into the culture barrel 1 through the carbon dioxide inlet pipe 3, with an aeration flow rate of 11 L / h. A pressure detection device is provided inside the barrel. When there are too many harmful gases inside the barrel, the pressure inside the barrel will increase. After reaching the set threshold, the one-way valve on the exhaust pipe 4 is opened, and the waste gas inside the barrel is discharged through the exhaust pipe 4 by using a power device such as an air pump. When the pressure inside the barrel does not reach the set threshold range, the one-way valve on the exhaust pipe 4 is closed. The corresponding valves on the culture barrel 1 can be manually adjusted or uniformly controlled by the main controller for automatic regulation. During the culture process, the feed pipe 5 is closed, the exhaust pipe 4 is not connected to the outside world in a non-exhaust state, and the two inlet pipes 5 are in the air intake (filtered air and carbon dioxide) state and are not directly connected to the external environment. Therefore, the inside of the culture barrel 1 is in a relatively sealed state to prevent external impurities from falling into the barrel body and polluting the culture environment.

[0069] Preferably, a gas distribution device is provided at the air outlet ends of the air inlet pipe 2 and the carbon dioxide inlet pipe 3 to disperse the gas input into the barrel, improve the uniformity of gas distribution, and thus help to improve the gas utilization rate and the cultivation effect.

[0070] During the culture process, continuously injecting air into the culture barrel 1 can mainly keep the protocorms suspended in the liquid culture medium in a state of turning, prevent the protocorms from agglomerating in the culture medium and causing their growth to be inhibited, maintain the uniformity of the suspension culture, and improve the nutrient absorption rate; and continuously introducing fresh air during the culture process can take away some waste gas produced by plant metabolism, which can prevent the local gas concentration imbalance from affecting cell growth and ensure the cleanliness of the culture environment; in addition, after the air is introduced, the dissolved oxygen concentration in the liquid culture medium can be increased, providing the necessary conditions for the aerobic respiration of the protocorms, thereby providing energy for the absorption of nutrients by Dendrobium officinale. So in general, continuously injecting fresh air during the culture process can maintain the respiration, gas exchange, metabolic balance of the protocorms and the dispersion state of the protocorms, thereby improving the nutrient absorption rate of the protocorms.

[0071] In addition, although the injected air contains a certain amount of carbon dioxide, it is not efficient to replenish carbon dioxide into a relatively closed barrel by simply filling it with air. Injecting carbon dioxide into the barrel while injecting air can make up for the problem of insufficient carbon dioxide, ensure an adequate supply of raw materials required for photosynthesis, promote the photosynthesis efficiency of the protocorm and optimize the metabolic environment. The increase in carbon dioxide concentration in the barrel can significantly increase the photosynthesis rate of the protocorm and promote the plant to synthesize more sugars and organic matter, thereby accelerating the growth rate of Dendrobium officinale shoots and enhancing biomass accumulation.

[0072] In addition, the additional addition of carbon dioxide during the tissue culture process can also replace part of the carbon source, breaking the limitations of the traditional culture method that mainly relies on sugar as a carbon source in terms of high cost, difficulty in further improving efficiency, and high pollution rate, and helping to successfully transform into a CO2 weak sugar mixotrophic model, that is, it is not to completely replace the carbon source with CO2 to establish a cultivation model that completely changes from heterotrophy to autotrophy, but to use CO2 to replace part of the carbon source for cultivation. A new weak sugar mixotrophic model that has both autotrophy and heterotrophy. The shortcomings of transitional reliance on sugar as a carbon source are high cost and easy to cause microbial contamination problems. The present application injects an appropriate amount of CO2 gas into the culture barrel 1 during the culture process to replace part of the carbon source, thereby optimizing the carbon source supply. Then, by accurately adjusting environmental factors such as light, the tissue culture cost can be controlled and the quality of the young shoots of Dendrobium officinale can be effectively improved.

[0073] Specifically, the concentration of carbon dioxide in the barrel needs to be appropriately adjusted according to the light-dark cycle. Since the protocorms of Dendrobium officinale may have insufficient carbon dioxide absorption during the light period due to low stomatal opening, and absorb and store carbon dioxide through open stomata during the dark period for photosynthesis during the light period. Therefore, during the light period cultivation, the carbon dioxide concentration in the barrel can be appropriately increased to 600-800 ppm (the carbon dioxide concentration cannot reach an exact and fixed value due to changes in gas temperature and pressure, mainly serving as a reference for adjustment) to make up for the insufficient absorption efficiency during the light period; during the dark period, it can be reduced to 300-500 ppm, and the concentration is monitored by a sensor set in the barrel to maintain the basic metabolic requirements.

[0074] After about 30 days of cultivation, the induction process of Dendrobium officinale tender buds can be completed, and induced cormels can be obtained in this step.

[0075] S3. Transfer the cormels obtained in S2 to the tender bud proliferation liquid medium for proliferation culture. The inoculation density is 10 g / L. The medium is also placed in the culture barrel 1 described in step S2. The filling volume of the medium in the barrel is 8 L. When the amount of the medium is less than 6 L, replenishment is required, and the replenishment volume is 3 L. The culture temperature is 20-26 °C, the relative air humidity is 55-60%, the light time is 16 h / day, and the light intensity is 3000 Lux (the temperature and humidity fluctuations within the corresponding ranges have little impact. In principle, the temperature is slightly higher during the light period and slightly lower during the dark period); an air inlet pipe 2, a carbon dioxide inlet pipe 3, an exhaust pipe 4, and a feed pipe 5 that penetrate through the inside and outside of the barrel are also connected to the barrel. The corresponding operations are the same as those in step S2. The feed pipe 5 is used to inject cormels into the barrel. During the proliferation culture of Dendrobium officinale tender buds, filtered compressed air is injected into the culture barrel through the air inlet pipe 2, and the ventilation volume is 4 L / min, and the flow rate is 10 m / s; and carbon dioxide gas is synchronously injected into the culture barrel 1 through the carbon dioxide inlet pipe 3, and the ventilation flow rate is 13 L / h. The exhaust pipe 4 is used to discharge the waste gas in the barrel. After about 15 days of cultivation, Dendrobium officinale tender buds can be obtained. The photo of the Dendrobium officinale tender buds cultivated in this example is referred to Figure 1 。

[0076] Example 2

[0077] The difference between this example and Example 1 is only that the dosage of the elemental mother liquor used in preparing the tender bud induction liquid medium and the tender bud proliferation liquid medium is adjusted, and the other components remain unchanged, and the dosages of the natural extracts (potato extract and coconut water) and plant growth hormones (6-BA, NAA) used in the medium remain unchanged. The culture conditions and culture process used during specific cultivation are exactly the same.

[0078] The formula of the tender bud induction liquid medium used in this example is as follows:

[0079] Measure 350 mL of potato extract, supplement distilled water to 600 mL, and sequentially add 35 mL of coconut water, 1.5 mL of the first organic element mother liquor, 8 mL of the sulfur-nitrogen-phosphorus element mother liquor, 6 mL of the calcium salt mother liquor, 8 mL of the ammonium salt mother liquor, 1 mL of the trace element mother liquor, 6 mL of the first iron salt mother liquor, 1 mL of the 6-BA mother liquor, and 0.4 mL of the NAA mother liquor. Add 18 g of sucrose, stir and mix well, make up the volume to 1000 mL with distilled water, adjust the pH to 6.10 with a 4 M potassium hydroxide solution, and sterilize at 121 °C and 1.1 MPa for 21 minutes to obtain the shoot induction liquid medium.

[0080] The formula of the shoot proliferation liquid medium is as follows:

[0081] Measure 350 mL of potato extract, supplement distilled water to 600 mL, and sequentially add 35 mL of coconut water, 1.5 mL of the first organic element mother liquor, 8 mL of the sulfur-nitrogen-phosphorus element mother liquor, 10 mL of the calcium salt mother liquor, 6 mL of the ammonium salt mother liquor, 1 mL of the trace element mother liquor, 8 mL of the first iron salt mother liquor, 0.3 mL of the 6-BA mother liquor, 18 g of sucrose, and 6 g of edible glucose. Stir and mix well, make up the volume to 1000 mL with distilled water, and adjust the pH to 6.10 with a 4 M potassium hydroxide solution. Sterilize at 121 °C and 1.1 MPa for 21 minutes to obtain the shoot proliferation liquid medium.

[0082] Example 3

[0083] The difference between this example and Example 1 is only that the dosages of the natural extracts (potato extract and coconut water) used in preparing the shoot induction liquid medium and the shoot proliferation liquid medium are reduced, and the other components remain unchanged. The culture conditions and the culture process used in the specific culture are exactly the same.

[0084] The formula of the shoot induction liquid medium used in this example is as follows:

[0085] Take 150 mL of potato extract, supplement distilled water to 600 mL, and sequentially add 20 mL of coconut water, 2 mL of the first organic element mother liquor, 10 mL of the sulfur-nitrogen-phosphorus element mother liquor, 10 mL of the calcium salt mother liquor, 10 mL of the ammonium salt mother liquor, 1 mL of the trace element mother liquor, 10 mL of the first iron salt mother liquor, 1 mL of the 6-BA mother liquor, and 0.4 mL of the NAA mother liquor. Add 18 g of sucrose, stir and mix well, make up the volume to 1000 mL with distilled water, adjust the pH to 6.10 with a 4 M potassium hydroxide solution, and sterilize at 121 °C and 1.1 MPa for 21 minutes to obtain the shoot induction liquid medium.

[0086] The formula of the shoot proliferation liquid medium is as follows:

[0087] Measure 150 mL of potato extract, supplement with distilled water to 600 mL, and sequentially add 20 mL of coconut water, 2 mL of the first organic element mother liquor, 10 mL of the sulfur-nitrogen-phosphorus element mother liquor, 10 mL of the calcium salt mother liquor, 10 mL of the ammonium salt mother liquor, 1 mL of the trace element mother liquor, 10 mL of the first iron salt mother liquor, 0.3 mL of the 6-BA mother liquor, 7 g of sucrose, and 5 g of edible glucose. Stir and mix well, make up the volume to 1000 mL with distilled water, adjust the pH to 6.1, and sterilize at 121 °C and 1.1 MPa for 21 minutes to obtain the shoot proliferation liquid medium.

[0088] Example 4

[0089] The difference between this example and Example 1 is only that the dosages of the growth hormones (6-BA, NAA) used in preparing the shoot induction liquid medium and the shoot proliferation liquid medium are reduced, and the other components remain unchanged. The culture conditions and the culture process used during the specific culture are exactly the same.

[0090] The formula of the shoot induction liquid medium used in this example is as follows:

[0091] Take 350 mL of potato extract, supplement with distilled water to 600 mL, and sequentially add 35 mL of coconut water, 2 mL of the first organic element mother liquor, 10 mL of the sulfur-nitrogen-phosphorus element mother liquor, 10 mL of the calcium salt mother liquor, 10 mL of the ammonium salt mother liquor, 1 mL of the trace element mother liquor, 10 mL of the first iron salt mother liquor, 0.2 mL of the 6-BA mother liquor, and 0.2 mL of the NAA mother liquor. Add 18 g of sucrose, stir and mix well, make up the volume to 1000 mL with distilled water, adjust the pH to 6.10 with a 4 M potassium hydroxide solution, and sterilize at 121 °C and 1.1 MPa for 21 minutes to obtain the shoot induction liquid medium.

[0092] The formula of the shoot proliferation liquid medium is as follows:

[0093] Measure 350 mL of potato extract, supplement with distilled water to 600 mL, and sequentially add 35 mL of coconut water, 2 mL of the first organic element mother liquor, 10 mL of the sulfur-nitrogen-phosphorus element mother liquor, 10 mL of the calcium salt mother liquor, 10 mL of the ammonium salt mother liquor, 1 mL of the trace element mother liquor, 10 mL of the first iron salt mother liquor, 0.1 mL of the 6-BA mother liquor, 7 g of sucrose, and 5 g of edible glucose. Stir and mix well, make up the volume to 1000 mL with distilled water, adjust the pH to 6.1, and sterilize at 121 °C and 1.1 MPa for 21 minutes to obtain the shoot proliferation liquid medium.

[0094] Comparative Example 1

[0095] The difference between this comparative example and Example 1 is that during the stages of inducing tender buds and proliferating tender buds from the protocorms of Dendrobium officinale, air and carbon dioxide are not continuously introduced into the culture tank for auxiliary culture, and other culture conditions and the culture media used in different stages are exactly the same.

[0096] Comparative Example 2

[0097] The difference between this comparative example and Example 1 is that during the stages of inducing tender buds and proliferating tender buds from the protocorms of Dendrobium officinale, only air is continuously introduced into the culture tank for auxiliary culture (i.e., carbon dioxide is not additionally introduced for auxiliary culture during the culture process), and other culture conditions and the culture media used are the same.

[0098] Comparative Example 3

[0099] The difference between this comparative example and Example 1 is that during the stages of inducing tender buds and proliferating tender buds of Dendrobium officinale, only air is continuously introduced into the culture tank for auxiliary culture, and the sugar content in the culture medium is increased, and other culture conditions are exactly the same.

[0100] The formula of the liquid medium for inducing tender buds is as follows:

[0101] Measure 350 mL of potato extract, supplement distilled water to 600 mL, and sequentially add 35 mL of coconut water (if coconut water produced by General Beverage Company, Thailand, the same below), 2 mL of the first organic element mother liquor, 10 mL of the sulfur-nitrogen-phosphorus element mother liquor, 10 mL of the calcium salt mother liquor, 10 mL of the ammonium salt mother liquor, 1 mL of the trace element mother liquor, 10 mL of the first iron salt mother liquor, 1 mL of the 6-BA mother liquor, and 0.4 mL of the NAA mother liquor. Add 30 g of sucrose, stir and mix evenly, make up the volume to 1000 mL with distilled water, adjust the pH of the solution to 6.10, and sterilize at 121 °C and 1.1 MPa pressure for 21 minutes to obtain the liquid medium for inducing tender buds. The pH of the culture medium will decrease to 5.6 - 5.8 after sterilization.

[0102] The formula of the liquid medium for proliferating tender buds is as follows:

[0103] Measure 350 mL of potato extract, supplement distilled water to 600 mL, and sequentially add 35 mL of coconut water, 2 mL of the first organic element mother liquor, 10 mL of the sulfur-nitrogen-phosphorus element mother liquor, 10 mL of the calcium salt mother liquor, 10 mL of the ammonium salt mother liquor, 1 mL of the trace element mother liquor, 10 mL of the first iron salt mother liquor, 0.3 mL of the 6-BA mother liquor, 20 g of sucrose, and 10 g of edible glucose. Stir and mix evenly, make up the volume to 1000 mL with distilled water, adjust the pH of the solution to 6.10, and sterilize at 121 °C and 1.1 MPa pressure for 21 minutes to obtain the liquid medium for proliferating tender buds.

[0104] Comparative Example 4

[0105] The difference between this comparative example and Example 1 is that the liquid medium for inducing tender buds and the liquid medium for proliferating tender buds used in this comparative example are both conventional MS media. The culture conditions and the culture process used during the specific culture are exactly the same as those in Example 1 (i.e., air and carbon dioxide need to be continuously introduced for auxiliary culture during the induction and proliferation stages of Dendrobium officinale tender buds).

[0106] MS medium ( / L): 1.65 g ammonium nitrate (NH4NO3), 1.9 g potassium nitrate (KNO3), 0.17 g potassium dihydrogen phosphate (KH2PO4), 0.37 g magnesium sulfate (MgSO4·7H2O), 0.44 g calcium chloride (CaCl2·2H2O), 6.2 mg boric acid (H3BO3), 22.3 mg manganese sulfate (MnSO4·4H2O), 8.6 mg zinc sulfate (ZnSO4·7H2O), 0.83 mg potassium iodide (KI), 0.25 mg sodium molybdate (Na2MoO4·2H2O), 0.025 mg copper sulfate (CuSO4·5H2O), 0.025 mg cobalt chloride (CoCl2·6H2O), 27.8 mg ferrous sulfate (FeSO4·7H2O), 37.2 mg disodium ethylenediaminetetraacetate (Na2-EDTA), 100 mg inositol, 0.5 mg nicotinic acid, 0.1 mg thiamine hydrochloride, 0.1 mg pyridoxine hydrochloride, 2 mg glycine, 18 g sucrose, pH: 5.7 - 5.8.

[0107] Comparative Example 5

[0108] The difference between this comparative example and Comparative Example 4 is only that air and carbon dioxide are not introduced for auxiliary culture during the culture in the induction and proliferation stages of Dendrobium officinale tender buds in this comparative example.

[0109] The culture effects of Examples 1 - 4 and Comparative Examples 1 - 5 were tested. The main tests were the induction rate of Dendrobium officinale tender buds, the proliferation coefficient, the polysaccharide content, the amino acid content, and the leaching content in the Dendrobium officinale tender buds. In addition, the number of days for the proliferation culture of Dendrobium officinale tender buds was also counted (the induction period of Dendrobium officinale tender buds in different examples and comparative examples was uniformly 30 days).

[0110] 1) Determination of the induction rate:

[0111] Using the protocorm of Dendrobium officinale as the explant, after culturing for a certain period of time, the germination number and growth situation of adventitious buds were counted, and the induction rate of tender buds and the average number of buds were calculated.

[0112] Induction rate = number of explants that induced cormels / total number of explants × 100%;

[0113] Average number of buds = total number of germinated buds / total number of explants.

[0114] 2) Determination of the proliferation coefficient:

[0115] Select healthy small bulbs of Dendrobium officinale as explants, regularly observe the growth of young shoots, and count the number of shoots after proliferation.

[0116] Proliferation coefficient = number of shoots after proliferation / number of initially inoculated shoots.

[0117] 3) Polysaccharide determination

[0118] It is carried out by the "Ultraviolet-Visible Spectrophotometry" in General Rule 0401 of Part IV of the Pharmacopoeia of the People's Republic of China (2020 Edition).

[0119] 4) Amino acid detection: It is carried out in accordance with GB 5009.124-2016 "National Food Safety Standard Determination of Amino Acids in Foods".

[0120] 5) Determination of extractives: It is carried out by General Rule 2201 of Part IV of the Pharmacopoeia of the People's Republic of China (2020 Edition). The specific test methods are as follows:

[0121] 1. Method for determining water-soluble extractives: Crush the test sample, sieve it through a No. 2 sieve, take about 4 g of the test sample, place it in a conical flask of 250 - 300 mL, add 100 mL of water, tightly stopper it, soak it in the cold, shake it constantly within the first 6 hours, then let it stand for 18 h, filter, measure 20 mL of the filtrate, place it in a dry evaporating dish, evaporate it to dryness in a water bath, dry it at 105 °C for 3 h, cool it for 30 min, and accurately weigh it. Unless otherwise specified, calculate the content (%) of water-soluble extractives in the test sample based on the dried product.

[0122] 2. Method for determining alcohol-soluble extractives: Determine it according to the method for determining water-soluble extractives, using ethanol of a specified concentration instead of water as the test solvent.

[0123] 3. Method for determining volatile ether extractives: Take 2 - 5 g of the test sample (sieved through a No. 4 sieve), dry it in a phosphorus pentoxide desiccator for 12 h, place it in a Soxhlet extractor, add an appropriate amount of ether, unless otherwise specified, heat and reflux for 8 h, take the ether solution, place it in a dry evaporating dish that has been dried to a constant weight, let it stand, volatilize the ether, place the residue in a phosphorus pentoxide desiccator and dry it for 18 h, accurately weigh it, slowly heat it to 105 °C, and dry it to a constant weight at 105 °C. The weight loss is the weight of the volatile ether extractives.

[0124] In Examples 1 - 4 and Comparative Examples 1 - 4, tissue culture of young shoots of Dendrobium officinale was carried out using different culture media or culture conditions, and the induction rate of young shoots, proliferation coefficient, contents of bioactive components polysaccharides and amino acids, extractive content, and the time used in the young shoot proliferation culture stage were measured under different conditions to evaluate the influence of different culture conditions on the culture results. The specific results are shown in Table 1.

[0125] Table 1. Influence of Different Culture Conditions on Tissue Culture of Young Shoots of Dendrobium officinale

[0126]

[0127] Analyze the data in the table: It can be seen from the data of Examples 1, 3 - 4 that the two natural extracts, potato extract and coconut water, have a certain promoting effect on the induction and proliferation culture of Dendrobium officinale young shoots. And appropriate concentrations of 6 - BA and NAA also have a promoting effect on the proliferation of protocorms. When the amount of natural extract or growth hormone in the culture medium is reduced, the induction rate and proliferation rate of Dendrobium officinale young shoots in the tissue culture process will also decrease to a certain extent.

[0128] It can be seen from the test data of Examples 1 - 2 that appropriately adjusting the amount of some stock solutions in the culture medium, that is, appropriately adjusting the amount of different elements in the culture medium, has not a very significant impact on the culture effect.

[0129] It can be seen from the data of Example 1 and Comparative Examples 1 - 2 that during the tissue culture of Dendrobium officinale young shoots, by continuously introducing a certain amount of air and carbon dioxide into the culture tank, the induction rate and proliferation rate of Dendrobium officinale young shoots can be improved, and it can also promote the increase of the content of active ingredients such as polysaccharides and amino acids in Dendrobium officinale young shoots to a certain extent. And compared with the scheme of only introducing air, additionally introducing carbon dioxide for co - culture also has an obvious impact on improving the cultivation effect.

[0130] Observing the test data of Comparative Examples 2 - 3, it can be seen that on the premise of not additionally introducing carbon dioxide gas for co - culture during the culture process, when the amount of sugar used as a carbon source in the induction and proliferation liquid medium of Dendrobium officinale young shoots is increased, the cultivation effect will be improved to a certain extent. However, compared with the scheme of co - culture based on a low - sugar medium and using carbon dioxide gas disclosed in Example 1, there is still a relatively obvious gap in the effect. This shows that by replacing part of the carbon source with CO2 gas, optimizing the ratio of macronutrients such as nitrogen, phosphorus, and potassium and trace elements in the culture medium, and then combining with the precise control of the protocorm development environment, the culture energy efficiency can be effectively improved on the basis of reducing sugar, achieving the goals of reducing tissue culture costs, significantly improving the quality and yield of Dendrobium officinale young stems. The proposed weak - sugar co - cultivation micro - propagation technology using CO2 to replace part of the carbon source in this application is scientifically feasible.

[0131] After increasing the amount of sucrose in the young shoot induction medium, it is found that under the condition of co - culture with carbon dioxide introduction, when the amount of sucrose in the culture medium is higher than 20 g, it has little impact on the cultivation effect and will not produce an obvious improvement effect; while when the amount of sucrose used in the young shoot induction medium is reduced to less than 10 g, it has an obvious impact on the induction effect of Dendrobium officinale young shoots, and the young shoot induction is inhibited. It is more reasonable to control the amount of sucrose in the medium within the range of 15 - 20 g during the induction process, preferably 18 g.

[0132] The sugar dosage in the liquid medium for the proliferation of tender buds was also adjusted. It was also found that when the dosages of sucrose and glucose were too high, there was little difference in the proliferation effect, but when they were too low, it would cause inhibition. Considering the perspective of not affecting the effect and saving costs, it was more reasonable to add 5 - 10 g of sucrose and 3 - 5 g of edible glucose, and 7 g of sucrose and 5 g of edible glucose were preferred.

[0133] It can also be seen from the above content that the weak - sugar co - cultivation idea proposed in this application is reasonable and feasible. Combining the co - cultivation with CO₂ on the basis of weak sugar can significantly improve the efficiency on the premise of cost reduction.

[0134] From the data of Comparative Examples 4 and 5, it can be further seen that during the tissue culture of Dendrobium officinale tender buds, introducing a certain amount of air and carbon dioxide into the culture barrel can significantly improve the seedling - raising effect and the content of active ingredients in Dendrobium officinale tender buds. Comparing the data of Example 1 and Comparative Example 4, it can be seen that the cultivation effect of the medium disclosed in this application is significantly improved compared with the basic MS medium. It not only helps to improve the induction rate and proliferation coefficient of Dendrobium officinale tender buds, but also increases the content of active ingredients in Dendrobium officinale tender buds, fully indicating that the medium used in Example 1 has a good growth - promoting effect and can maintain the stable traits of Dendrobium officinale tender buds.

[0135] In addition, it was found through testing that compared with the culture conditions of Comparative Example 5, when cultivating Dendrobium officinale under the culture conditions described in Example 1, the yield of Dendrobium officinale tender buds per unit area increased from the traditional 3200 g / barrel to 4300 g / barrel (see Table 2), further indicating that this application achieved the purpose of promoting the differentiation, proliferation and growth and development of protocorms and Dendrobium officinale tender buds by optimizing environmental factors and nutrient supply.

[0136] Table 2. Comparison of basic index parameters between Example 1 and Comparative Example 5

[0137]

[0138] In addition, after statistically analyzing the time used in the proliferation culture stage of Dendrobium officinale tender buds under different culture conditions, it was found that using the medium disclosed in Example 1 of this application for the induction and proliferation culture of Dendrobium officinale tender buds and continuously introducing air and carbon dioxide for auxiliary co - cultivation during the culture process not only did not damage the traits of the cultivated Dendrobium officinale tender buds (the content of active ingredients in the tender buds was stable), but also significantly shortened the time required for the proliferation culture stage of Dendrobium officinale tender buds. Furthermore, it helped to shorten the entire cultivation cycle, significantly increase the yield of Dendrobium officinale in the marketization stage, reduce the cultivation cost, quickly respond to market demand, enhance the market competitiveness of enterprises and increase enterprise revenue, ultimately providing a core driving force for the efficient development of the Dendrobium officinale industry.

[0139] Application Example

[0140] This application example is mainly used to verify whether the tender buds of Dendrobium officinale cultivated under the best cultivation conditions have an improvement effect on pain. Specifically, the acetic acid writhing test is used for research. The acetic acid writhing test in mice is a chemical stimulation pain method, which is an inflammatory pain manifested by writhing reaction caused by intraperitoneal injection of a certain amount of acetic acid to stimulate the peritoneum of mice.

[0141] Take the tender buds of Dendrobium officinale cultivated in Example 1, dry them and grind them into dry powder, and sieve them through a 80-mesh sieve for standby.

[0142] Four-week-old male KM mice were randomly divided into a blank control group (Con), a low-dose administration group of Dendrobium officinale tender buds (Low), and a high-dose administration group of Dendrobium officinale tender buds (High). The blank control group was intragastrically administered carboxymethyl cellulose sodium (CMC-Na). The samples used in the experimental groups were prepared by suspending 99.5% of the dry powder of Dendrobium officinale tender buds and 0.5% of carboxymethyl cellulose sodium. The low-dose group was intragastrically administered according to the standard of 1170 mg / kg / d, and the high-dose group was intragastrically administered according to the standard of 3510 mg / kg / d. The mice were intragastrically administered once a day at an equal volume of 0.2 mL / 10 g body weight for 14 consecutive days.

[0143] On the 14th day of drug administration, the acetic acid writhing test was carried out. That is, 30 minutes after the last drug administration, each mouse was intraperitoneally injected with 0.3 mL of 0.6% acetic acid solution preheated in a 37°C water bath, and the number of writhing reactions occurring within 20 minutes was observed and recorded, and the analgesia rate was calculated. The manifestations of the writhing reaction were abdominal contraction, body distortion, hind limb extension, and creeping, etc. The calculation method of the analgesia rate was:

[0144] Analgesia rate (%) = (number of writhing times in the control group - number of writhing times in the drug administration group) / number of writhing times in the control group × 100%.

[0145] For the experimental results, see Table 3, Table 4 and Figure 4 Judging from the data in the two tables, compared with the blank control group, the number of writhing times in the low- and high-dose groups of Dendrobium officinale tender buds was significantly reduced, and the analgesia rates were higher than those of the blank control group. Among them, the high-dose group of Dendrobium officinale tender buds showed the least number of writhing times, and the analgesia rate reached 77.58%, and the analgesic effect was significant.

[0146] Table 3. Writhing latency and number of writhing times of mice in each group

[0147]

[0148]

[0149] Note: " / " means that no writhing reaction occurred, so the latency was not recorded.

[0150] Table 4. Average number of writhing times and analgesia percentage of mice in each group

[0151]

[0152] Note: The results are expressed as Mean ± SE.M (n = 11 - 12). *** indicates significant difference from the control group with P < 0.001, and ** indicates significant difference from the control group with P < 0.01.

[0153] In this study, the acetic acid writhing test was used to investigate the improvement effect of the tender buds of Dendrobium officinale on the pain of mice. The pain writhing response that occurred in the experiment conformed to the experimental methodology design. After the action of the tender buds of Dendrobium officinale cultivated by this application, the number of writhing times of the mice decreased significantly, and the latency was prolonged. The analgesic rate of the high-dose group (3510 mg / kg / d) of the tender buds of Dendrobium officinale was greater than 50%, proving that the tender buds of Dendrobium officinale at this dose had a good analgesic effect.

[0154] The above shows and describes the basic principles, main features and advantages of the present invention. However, the above are only specific embodiments of the present invention, and the technical features of the present invention are not limited thereto. Any other embodiments obtained by those skilled in the art without departing from the technical solution of the present invention should be covered within the scope of the present invention.

Claims

1. A method for cultivating tender buds of Dendrobium officinale under weak sugar and aerated conditions, characterized in that, It includes the following steps: 1) Inoculate the disinfected Dendrobium officinale seeds into the protocorm induction medium to obtain Dendrobium officinale protocorms; 2) Inoculate the Dendrobium officinale protocorms into the weak sugar liquid medium for shoot induction. During the induction culture process, continuously introduce air and carbon dioxide gas into the medium to obtain induced bulblets; 3) Inoculate the bulblets obtained in the previous step into the weak sugar liquid medium for shoot proliferation. During the proliferation culture process, continuously introduce air and carbon dioxide gas into the medium to obtain Dendrobium officinale shoots.

2. The method for cultivating Dendrobium officinale young shoots under weak sugar and aerated conditions as described in claim 1, characterized in that, In step 2), the inoculation amount of protocorms in the medium is 12 - 14 g / L, the temperature during the shoot induction culture process is 21 - 26 °C, the relative air humidity is 45 - 55%, and the light duration is 12 - 14 h / day.

3. The method for cultivating tender buds of Dendrobium officinale under weak sugar and aerated conditions as described in claim 1, characterized in that, In step 2), the compressed air is introduced into the medium during the induction culture process, the ventilation volume is 3 - 4 L / min, and the flow rate is 10 - 12 m / s; the concentration of carbon dioxide in the culture vessel is adjusted according to the light - dark cycle. The concentration of carbon dioxide in the vessel during the light period is 600 - 800 ppm, and it is adjusted to 300 - 500 ppm during the dark period.

4. The method for cultivating tender buds of Dendrobium officinale under weak sugar and aerated conditions as described in claim 1, characterized in that, In step 3), the inoculation amount of bulblets in the medium is 10 - 12 g / L, the temperature during the shoot proliferation culture process is 20 - 26 °C, the relative air humidity is 55 - 60%, and the light duration is 14 - 16 h / day.

5. The method for cultivating tender buds of Dendrobium officinale under weak sugar and aeration conditions as described in claim 1, characterized in that, In step 3), the compressed air is introduced into the medium, the ventilation volume is 4 - 5 L / min, and the flow rate is 10 - 12 m / s; the concentration of carbon dioxide in the culture vessel is adjusted according to the light - dark cycle. The concentration of carbon dioxide in the vessel during the light period is 600 - 800 ppm, and it is adjusted to 300 - 500 ppm during the dark period.

6. The method for cultivating tender buds of Dendrobium officinale under weak sugar and aerated conditions as described in claim 1, characterized in that, In step 2), the preparation method of the weak sugar liquid medium for shoot induction is as follows: Measure 150 - 350 mL of potato extract, supplement distilled water to 500 - 700 mL, and successively add 30 - 40 mL of coconut water, 1.5 - 2.5 mL of the first organic element mother liquor, 5 - 15 mL of the sulfur - nitrate - phosphorus element mother liquor, 5 - 15 mL of the calcium salt mother liquor, 5 - 15 mL of the ammonium salt mother liquor, 0.5 - 1.5 mL of the trace element mother liquor, 5 - 15 mL of the first iron salt mother liquor, 0.2 - 1.5 mL of the 6 - BA mother liquor, and 0.2 - 0.6 mL of the NAA mother liquor. Add 15 - 20 g of sucrose, stir and mix evenly, make up the volume to 1000 mL with distilled water, adjust the pH of the solution to 6.10, and sterilize at 121 °C for 21 minutes.

7. The method for cultivating tender buds of Dendrobium officinale under weak sugar and aerated conditions according to claim 6, characterized in that, In step 3), the preparation method of the weak sugar liquid medium for shoot proliferation is as follows: Measure 150 - 350 mL of potato extract, supplement distilled water to 500 - 700 mL, and sequentially add 30 - 40 mL of coconut water, 1.5 - 2.5 mL of the first organic element mother liquor, 5 - 15 mL of the sulfur-nitrogen-phosphorus element mother liquor, 5 - 15 mL of the calcium salt mother liquor, 5 - 15 mL of the ammonium salt mother liquor, 0.5 - 1.5 mL of the trace element mother liquor, 5 - 15 mL of the first iron salt mother liquor, 0.15 - 0.45 mL of the 6-BA mother liquor, 5 - 10 g of sucrose and 3 - 5 g of edible glucose, stir and mix evenly, make up the volume to 1000 mL with distilled water, adjust the pH of the solution to 6.10, and sterilize at 121 °C for 21 minutes.

8. The method for cultivating tender buds of Dendrobium officinale under weak sugar and aeration conditions according to claim 7, characterized in that, The preparation methods of different element mother liquors and potato extract are as follows: Each liter of the first organic element mother liquor contains: 1 g of glycine, 0.25 g of pyridoxine hydrochloride, 0.05 g of thiamine hydrochloride, 0.25 g of nicotinic acid, 50 g of inositol, and the balance is deionized water; Each liter of the sulfur-nitrogen-phosphorus element mother liquor contains: 37 g of magnesium sulfate, 190 g of potassium nitrate, 17 g of potassium dihydrogen phosphate, and the balance is deionized water; Each liter of the calcium salt mother liquor contains: 44 g of calcium chloride, 28 g of calcium nitrate, and the balance is deionized water; Each liter of the ammonium salt mother liquor contains: 48.5 g of ammonium sulfate, and the balance is deionized water; Each liter of the first iron salt mother liquor contains: 3.45 g of ferrous sulfate, 3.73 g of disodium ethylenediaminetetraacetate, and the balance is deionized water; Each liter of the trace element mother liquor contains: 16.9 g of manganese sulfate, 8.6 g of zinc sulfate, 6.2 g of boric acid, 0.83 g of potassium iodide, 0.25 g of sodium molybdate, 0.025 g of copper sulfate, 0.025 g of cobalt chloride, and the balance is deionized water; 6-BA mother liquor: 80 - 120 mg of 6-BA is dissolved in 5 - 15 mL of HCl solution with a concentration of 0.5 - 1.5 mol / L, and the volume is made up to 50 - 150 mL with deionized water; NAA mother liquor: 30 - 60 mg of naphthylacetic acid is dissolved in 15 - 25 mL of ethanol solution, and the volume is made up to 50 - 110 mL with deionized water; Potato extract: Wash and peel the potatoes and cut them into slices. Put 150 - 200 g of potato slices into 350 - 450 mL of distilled water and cook for 20 - 30 min, then filter and collect the filtrate.

9. The method for cultivating tender buds of Dendrobium officinale under weak sugar and aerated conditions according to claim 8, characterized in that, The preparation method of the weak sugar liquid medium for shoot induction is as follows: Measure 350 mL of potato extract, supplement distilled water to 600 mL, and sequentially add 35 mL of coconut water, 2 mL of the first organic element mother liquor, 10 mL of the sulfur-nitrogen-phosphorus element mother liquor, 10 mL of the calcium salt mother liquor, 10 mL of the ammonium salt mother liquor, 1 mL of the trace element mother liquor, 10 mL of the first iron salt mother liquor, 1 mL of the 6-BA mother liquor and 0.4 mL of the NAA mother liquor, add 18 g of sucrose, stir and mix evenly, make up the volume to 1000 mL with distilled water, adjust the pH of the solution and then sterilize; The preparation method of the weak sugar liquid medium for shoot proliferation is as follows: Measure 350 mL of potato extract, supplement distilled water to 600 mL, and sequentially add 35 mL of coconut water, 2 mL of the first organic element mother liquor, 10 mL of the sulfur-nitrogen-phosphorus element mother liquor, 10 mL of the calcium salt mother liquor, 10 mL of the ammonium salt mother liquor, 1 mL of the trace element mother liquor, 10 mL of the first iron salt mother liquor, 0.3 mL of the 6-BA mother liquor, 7 g of sucrose and 5 g of edible glucose, stir and mix evenly, make up the volume to 1000 mL with distilled water, adjust the pH of the solution and then sterilize it.

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