Dendrobium officinale culture medium and application thereof

Abstract

The invention relates to the technical field of plant tissue culture, and particularly discloses a dendrobium officinale culture medium and application thereof.The dendrobium officinale culture medium is prepared from MS basic mother liquor, cane sugar, casein hydrolysate, mashed bananas, potato extract, coconut shell powder, plant hormones (6-benayl aminopurine, naphthylacetic acid and kinetin), rare earth element compounds, activated carbon and water in specific parts. The rare earth element compound is formed by mixing lanthanum nitrate, cerium nitrate and gadolinium nitrate according to the ratio of 2: 1: 3, when the rare earth element compound is added, the lanthanum nitrate, the cerium nitrate and the gadolinium nitrate are dissolved in water on a sterile operation table and filtered and sterilized through a 0.22-micron filter membrane, meanwhile, the culture medium without rare earth is sterilized through high-pressure steam, and after the culture medium is cooled to 45-50 DEG C, the solution is injected through a quantitative pipetting device and mixed to be uniform. The culture medium is accurately blended according to the unique growth habit of dendrobium officinale, nutrients are comprehensive and balanced, the requirements of all stages can be fully met, the scientific addition of rare earth elements can promote photosynthesis and accumulation of effective components, and a good substrate is provided for artificial cultivation of dendrobium officinale in cooperation with rigorous sterilization operation.

Description

Technical Field

[0001] The present invention relates to the technical field of plant tissue culture, and particularly to a culture medium for Dendrobium officinale and its application. Background Art

[0002] As a precious medicinal plant, Dendrobium officinale has extremely high application value in the field of traditional Chinese medicine. It is rich in various effective components such as dendrobine and polysaccharides, and has many benefits to human health, such as enhancing immunity, nourishing yin and clearing heat, etc. The market demand has been continuously rising. However, the natural growth environment of Dendrobium officinale is harsh, and it is mostly distributed on the rocky cliffs and tree trunks in semi-shady and humid mountainous areas. With over-exploitation, the wild resources are on the verge of exhaustion, and artificial cultivation has become the key way to meet the market demand and protect wild resources.

[0003] Traditional cultivation of Dendrobium officinale mostly refers to the culture medium formula of ordinary plants. However, Dendrobium officinale has unique growth habits and nutritional requirements, and ordinary culture media are difficult to provide the precise nutrients required at each stage of its growth and development, resulting in problems such as slow plant growth, low survival rate, and insufficient accumulation of medicinal components. For example, in the seedling stage, sufficient and easily absorbable nitrogen sources are needed to promote the growth of stems and leaves, while some existing culture media cannot meet this efficiently; in the adult plant stage, the demand for phosphorus, potassium and specific trace elements changes, and if the supply cannot be adapted, it will directly affect flowering, fruiting and the synthesis of effective components.

[0004] In addition, the sterilization link of the culture medium is also crucial. Inappropriate sterilization methods may damage the nutritional components of the culture medium, and the growth of miscellaneous bacteria will compete with Dendrobium officinale for nutrients and cause diseases, seriously hindering its healthy growth. Traditional single high-temperature and high-pressure sterilization is likely to cause caramelization of nutrients such as sugars, changing the physical and chemical properties of the culture medium; if the sterilization is not thorough, the residues of bacteria and fungal spores will multiply in large numbers during the subsequent cultivation process, endangering the plants.

[0005] Furthermore, regarding the application of rare earth elements, they have been less involved in plant culture media in the past, especially for plants like Dendrobium officinale that are sensitive to the environment and have fine nutritional requirements. Although rare earth elements have unique regulatory effects in plant physiological processes, such as promoting photosynthesis and enhancing stress resistance, due to their complex chemical properties, improper addition methods are likely to cause precipitation and aggregation, not only failing to exert their effects, but also being toxic to plant cells.

[0006] Generally speaking, the existing cultivation techniques of Dendrobium officinale need to be improved urgently. There is an urgent need for a special culture medium that can precisely meet the growth requirements of Dendrobium officinale, optimize the sterilization process, and rationally utilize special components such as rare earth elements, so as to promote the efficient and sustainable development of the artificial cultivation industry of Dendrobium officinale. Summary of the Invention

[0007] (1) Technical Problems to be Solved

[0008] In view of the deficiencies of the prior art, the present invention provides a Dendrobium officinale culture medium and its application, which solves the problems raised in the above-mentioned background art.

[0009] (II) Technical solution

[0010] In order to achieve the above object, the present invention discloses a special culture medium for Dendrobium officinale. The culture medium is composed of 50 - 60 parts of MS basal mother liquor, 8 - 12 parts of sucrose, 5 - 10 parts of hydrolyzed casein, 6 - 10 parts of banana puree, 4 - 8 parts of potato extract, 3 - 7 parts of coconut shell powder, 0.5 - 2 parts of 6 - benzylaminopurine, 0.3 - 1.5 parts of naphthaleneacetic acid, 0.1 - 1 part of kinetin, 4 - 12 parts of rare earth element compound, 3 - 8 parts of activated carbon, and 25 - 40 parts of water; the rare earth element compound is a mixture of lanthanum nitrate, cerium nitrate and gadolinium nitrate, and the mass ratio of lanthanum nitrate, cerium nitrate and gadolinium nitrate is 2:1:3; the rare earth element compound is added to the culture medium according to the following method: in a sterile operation table environment, lanthanum nitrate, cerium nitrate and gadolinium nitrate are dissolved in water to form a solution, which is filtered and sterilized through a 0.22 μm filter membrane. At the same time, the culture medium without the addition of rare earth element compound is sterilized by high - pressure steam. When the temperature of the culture medium drops to 45 - 50 °C, the solution is injected into the culture medium by a quantitative pipetting device and shaken well.

[0011] Preferably, the rare earth element compound is replaced by a mixture of praseodymium nitrate, yttrium nitrate and europium nitrate, and the mass ratio of praseodymium nitrate, yttrium nitrate and europium nitrate is 3:2:1.

[0012] Preferably, the culture medium further contains 2 - 5 parts of seaweed extract, and the seaweed extract is prepared by the following steps: the dried seaweed is crushed and mixed with distilled water in a ratio of 1:10, extracted in a water bath at 60 °C for 3 hours, and the supernatant is centrifuged and concentrated to 1 / 5 of the original volume.

[0013] Preferably, the high - pressure steam sterilization adopts a staged sterilization process. In the first stage, it is sterilized at 121 °C for 15 minutes, cooled to 80 °C and maintained for 30 minutes, and then heated to 115 °C for 10 minutes.

[0014] Preferably, the activated carbon is pretreated, specifically, the activated carbon is soaked in 0.1 mol / L hydrochloric acid for 2 hours, washed to neutrality, dried and sieved through a 200 - mesh sieve.

[0015] Another object of the present invention is to provide an application of the special culture medium for Dendrobium officinale, which is used for callus induction of Dendrobium officinale, protocorm proliferation or synchronous rooting and strong seedling cultivation of in vitro regenerated plants.

[0016] (III) Beneficial technical effects

[0017] In terms of nutritional supply, its unique formula precisely adapts to each growth stage of Dendrobium officinale. The MS base mother liquor, combined with ingredients like sucrose and hydrolyzed casein, provides ample nitrogen and energy for rapid stem and leaf germination during the seedling stage, helping the seedlings thrive and improving initial survival rates. Natural organic ingredients like banana puree and potato extract, rich in vitamins and minerals, meet the plant's meticulous trace element needs throughout its growth process, ensuring healthy development and robust growth. This results in stronger plants, thicker stems, and richer, greener leaves, laying a solid foundation for subsequent flowering and fruiting.

[0018] The scientific application of rare earth element compounds is a major highlight. Lanthanum nitrate, cerium nitrate, and gadolinium nitrate (or praseodymium nitrate, yttrium nitrate, and europium nitrate) mixed in specific proportions and added under strict aseptic conditions effectively promotes photosynthesis in Dendrobium officinale. Rare earth elements can regulate chloroplast structure and function, increase photosynthetic pigment content, and significantly enhance the plant's photosynthetic efficiency, accumulating more organic matter for growth and the synthesis of medicinal ingredients. This significantly increases the content of active ingredients such as dendrobium alkaloids and polysaccharides, enhancing the quality of the medicinal material.

[0019] During the sterilization phase of culture medium preparation, an innovative, phased high-pressure steam sterilization process and activated carbon pretreatment complement each other. This phased sterilization process not only avoids excessive destruction of nutrients but also ensures the complete elimination of all bacteria and spores, creating a pure and sterile starting environment for Dendrobium officinale. Pre-treated with hydrochloric acid, the activated carbon, through soaking, washing, and screening, not only effectively absorbs harmful substances that may be generated during culture medium preparation and subsequent use, but also regulates the culture medium's air permeability, facilitating root respiration and nutrient absorption, reducing the incidence of root diseases, and further enhancing plant growth vitality.

[0020] The additionally added seaweed extract is rich in a variety of natural growth regulators and minerals, which can fully stimulate the growth potential of Dendrobium officinale, promote cell division and elongation, work synergistically with other ingredients, shorten the growth cycle, increase the yield per unit area, and provide strong support for the large-scale, high-quality artificial cultivation of Dendrobium officinale, promoting both economic and social benefits of related industries. DETAILED DESCRIPTION

[0021] To facilitate understanding of the present invention, the present invention will be described in more detail below. Preferred embodiments of the present invention are provided below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of the present invention.

[0022] Example 1

[0023] The preparation method of the special culture medium for Dendrobium officinale is as follows:

[0024] Raw material preparation: Weigh 55 parts of MS basal mother liquor, 10 parts of sucrose, 8 parts of hydrolyzed casein, 8 parts of banana puree, 6 parts of potato extract, 5 parts of coconut shell powder, 1.2 parts of 6-benzylaminopurine, 0.8 part of naphthylacetic acid, 0.6 part of kinetin, 2 parts of lanthanum nitrate, 1 part of cerium nitrate, 3 parts of gadolinium nitrate, 5 parts of activated carbon, and 35 parts of water by mass;

[0025] Culture medium preparation: Mix MS basal mother liquor, sucrose, hydrolyzed casein, banana puree, potato extract, coconut shell powder and water, heat to 60 °C and stir to dissolve, add activated carbon and then dispense into culture bottles;

[0026] Sterilization treatment: Adopt the high-pressure steam sterilization process, sterilize at 121 °C for 15 minutes in the first stage, cool to 80 °C and maintain for 30 minutes, then heat up to 115 °C and sterilize for 10 minutes;

[0027] Rare earth element addition: Dissolve lanthanum nitrate, cerium nitrate and gadolinium nitrate in 10 mL of sterile water, filter through a 0.22 μm filter membrane, and inject the solution into the medium when the medium cools to 48 °C, and shake well;

[0028] Dispensing and storage: Dispense into sterile culture containers and store in the dark at 4 °C after cooling.

[0029] Example 2

[0030] Raw material adjustment: Replace lanthanum nitrate, cerium nitrate and gadolinium nitrate in Example 1 with 3 parts of praseodymium nitrate, 2 parts of yttrium nitrate and 1 part of europium nitrate, and the other components are the same as in Example 1;

[0031] Preparation steps: The same as in Example 1, only adjust the types and proportions of rare earth elements.

[0032] Example 3

[0033] Add seaweed extract: Add 3 parts of seaweed extract on the basis of the formula in Example 1, and the preparation steps are as follows:

[0034] Seaweed extract preparation: Crush the dried seaweed and mix it with distilled water at a ratio of 1:10, extract in a water bath at 60 °C for 3 hours, centrifuge and take the supernatant and concentrate it to 1 / 5 of the original volume;

[0035] Culture medium preparation: Add the seaweed extract and other raw materials to the mixture synchronously, and the subsequent steps are the same as in Example 1.

[0036] Example 4

[0037] Staged sterilization optimization: Adopt the staged sterilization process, sterilize at 121 °C for 15 minutes in the first stage, cool to 80 °C and maintain for 30 minutes, then heat up to 115 °C and sterilize for 10 minutes;

[0038] Other steps: The same as in Example 1.

[0039] Comparative Example 1

[0040] Formula adjustment: omit the rare earth element compound, and the remaining components are the same as in Example 1;

[0041] Preparation steps: Same as Example 1.

[0042] Comparative Example 2

[0043] Sterilization process adjustment: conventional sterilization at 121°C for 30 minutes without staged sterilization;

[0044] The remaining steps are the same as those in Example 1.

[0045] Comparative Example 3

[0046] Activated carbon without pretreatment: activated carbon without hydrochloric acid treatment was used directly;

[0047] Preparation steps: Same as Example 1.

[0048] Comparison of experimental results:

[0049] The following are the effect data of cultivating Dendrobium officinale in the embodiment and the comparative example (cultivation period is 30 days):

[0050]

[0051]

[0052] Key group distribution ratio table:

[0053]

[0054] Implementation Instructions:

[0055] Rare earth element compounds are sterilized in stages and injected at low temperatures to effectively avoid high-temperature decomposition; the addition of seaweed extract further promotes callus differentiation and shortens the culture cycle;

[0056] The staged sterilization process reduces nutrient loss caused by activated carbon adsorption and improves sterilization efficiency.

[0057] Example 5

[0058] The preparation method of the special culture medium for Dendrobium officinale is as follows:

[0059] Raw material optimization: Weigh, by mass, 58 parts of MS base mother liquor, 9 parts of sucrose, 7 parts of hydrolyzed casein, 7 parts of banana puree, 5 parts of potato extract, 4 parts of coconut shell powder, 1.0 part of 6-benzylaminopurine, 0.6 parts of naphthaleneacetic acid, 0.4 parts of kinetin, 2.5 parts of lanthanum nitrate, 1.2 parts of cerium nitrate, 3.5 parts of gadolinium nitrate, 4 parts of activated carbon, and 32 parts of water;

[0060] Activated carbon pretreatment: Immerse the activated carbon in 0.1 mol / L hydrochloric acid solution for 2 hours, wash until neutral, dry, and pass through a 200-mesh sieve;

[0061] Mixing and sterilization: Mix and dissolve the raw materials except for the rare earth compounds, aliquot, and perform staged sterilization (121 °C / 15 minutes → 80 °C / 30 minutes → 115 °C / 10 minutes);

[0062] Injection of rare earth solution: Dissolve lanthanum nitrate, cerium nitrate, and gadolinium nitrate in proportion in 8 mL of sterile water, filter, and inject into the culture medium at 50 °C, then shake well to mix.

[0063] Example 6

[0064] Adjustment of high-concentration rare earth: Increase the total amount of rare earth element compounds in Example 1 to 12 parts (4.8 parts of lanthanum nitrate, 2.4 parts of cerium nitrate, 7.2 parts of gadolinium nitrate), and keep the other components unchanged;

[0065] Preparation process: The same as in Example 1, control the injection temperature of the rare earth solution at 47 °C.

[0066] Example 7

[0067] Application of composite additive: Add 4 parts of seaweed extract and 0.5 part of proline to the formula of Example 1 at the same time;

[0068] Preparation of seaweed extract: Extract according to the method of Example 3, concentrate, and add it to the mixture synchronously with proline;

[0069] Sterilization and aliquot: Adopt the staged sterilization process of Example 4.

[0070] Comparative example 4

[0071] No activated carbon: Omit the activated carbon component in Example 1;

[0072] Preparation steps: The same as in Example 1.

[0073] Comparative example 5

[0074] Disordered rare earth ratio: Adjust the mass ratio of lanthanum nitrate, cerium nitrate, and gadolinium nitrate to 1:1:1, and the total number of parts is 6 parts;

[0075] Preparation steps: The same as in Example 1.

[0076] Experimental effect extended comparison:

[0077] The following are the culture effect data of the new examples and comparative examples (culture period: 30 days):

[0078]

[0079] Sterilization process comparison table:

[0080]

[0081] Description of implementation details:

[0082] Function of rare earth elements: Lanthanum nitrate, cerium nitrate, and gadolinium nitrate are mixed in a ratio of 2:1:3, which can synergistically promote the synthesis of Dendrobium officinale polysaccharide. Although the high concentration of rare earth in Example 6 increases the proliferation coefficient, it may cause slight browning due to the increase in osmotic pressure;

[0083] Effect of composite additive: In Example 7, the combination of proline and seaweed extract significantly inhibits browning, and the chlorophyll content increases by 12%;

[0084] Necessity of activated carbon: The absence of activated carbon in Comparative Example 4 leads to a sharp increase in the pollution rate and browning rate, indicating that its functions of adsorbing toxins and stabilizing pH are irreplaceable;

[0085] Advantages of sterilization process: The staged sterilization controls the temperature gradient, reduces the degradation of thermosensitive substances (such as kinetin), and the nutrient retention rate is increased by 11.3% compared with conventional sterilization.

[0086] Note: In all examples, the pH value of the culture medium is adjusted to 5.8 - 6.2, and it needs to be used within 24 hours after dispensing to ensure the stability of the active ingredients.

[0087] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. All equivalent changes and improvements made within the scope of the present invention application shall still fall within the scope covered by the patent of the present invention.

Claims

1. A special culture medium for Dendrobium officinale, characterized in that: The culture medium is composed of 50 - 60 parts of MS basal mother liquor, 8 - 12 parts of sucrose, 5 - 10 parts of casein hydrolysate, 6 - 10 parts of banana puree, 4 - 8 parts of potato extract, 3 - 7 parts of coconut shell powder, 0.5 - 2 parts of 6 - benzylaminopurine, 0.3 - 1.5 parts of naphthaleneacetic acid, 0.1 - 1 part of kinetin, 4 - 12 parts of rare earth element compound, 3 - 8 parts of activated carbon, and 25 - 40 parts of water; the rare earth element compound is a mixture of lanthanum nitrate, cerium nitrate, and gadolinium nitrate, and the mass ratio of lanthanum nitrate, cerium nitrate, and gadolinium nitrate is 2:1:3; the rare earth element compound is added to the culture medium by the following method: in the environment of a sterile operating table, dissolve lanthanum nitrate, cerium nitrate, and gadolinium nitrate in water to form a solution, filter and sterilize it through a 0.22μm filter membrane, and at the same time, sterilize the culture medium without adding the rare earth element compound by high - pressure steam sterilization. When the temperature of the culture medium drops to 45 - 50°C, inject the solution into the culture medium using a quantitative pipetting device and mix it by shaking.

2. The special culture medium for Dendrobium officinale as claimed in claim 1, wherein: The rare earth element compound is replaced with a mixture of praseodymium nitrate, yttrium nitrate, and europium nitrate, and the mass ratio of praseodymium nitrate, yttrium nitrate, and europium nitrate is 3:2:

1.

3. The special culture medium for Dendrobium officinale according to claim 1 or 2, characterized in that: The culture medium further contains 2 - 5 parts of seaweed extract, and the seaweed extract is prepared by the following steps: crush the dried seaweed and mix it with distilled water in a ratio of 1:10, extract it in a water bath at 60°C for 3 hours, centrifuge it, and take the supernatant and concentrate it to 1 / 5 of the original volume.

4. The special culture medium for Dendrobium officinale according to claim 1, characterized in that: The high - pressure steam sterilization adopts a staged sterilization process. In the first stage, sterilize at 121°C for 15 minutes, cool to 80°C and maintain for 30 minutes, and then heat up to 115°C and sterilize for 10 minutes.

5. The special culture medium for Dendrobium officinale according to claim 1, wherein: The activated carbon is pretreated. Specifically, soak the activated carbon in 0.1mol / L hydrochloric acid for 2 hours, wash it until neutral, dry it, and sieve it through a 200 - mesh sieve.

6. Use of a special culture medium for Dendrobium officinale as described in any one of claims 1-5, characterized in that: The culture medium is used for the induction of Dendrobium officinale callus, the proliferation of protocorms, or the synchronous rooting and strong - seedling cultivation of in vitro regenerated plants.

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