Tissue culture and rapid propagation method for medicinal plants

Abstract

The invention discloses a medicinal plant tissue culture and rapid propagation method, which belongs to the technical field of plant tissue culture and is realized by three steps of efficient plant selection culture, intelligent intermittent immersion culture, sterile transplanting and rapid propagation. Through optimized culture medium components and an intelligent intermittent immersion system, the differentiation rate and rooting rate of plants are remarkably improved. For example, in an experiment of astragalus membranaceus, the differentiation rate and the rooting rate are increased by 25% and 29% respectively; in experiments of pinellia ternate, the two indexes are respectively improved by 22% and 26%. By adopting the method, the culture period is greatly shortened, and the tissue culture efficiency is improved. In the tissue culture of astragalus membranaceus and pinellia ternate, the culture periods are respectively shortened by 38% and 37%, so that rapid propagation becomes possible. The fresh weight of a single plant is remarkably increased by finely regulating and controlling the culture conditions such as the photoperiod, the temperature and the components of the nutrient solution. For example, the fresh weight of a single plant of astragalus membranaceus is increased by 50%, and the fresh weight of pinellia ternate is increased by 55%.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, and more specifically to a method for rapid propagation of medicinal plant tissue culture. Background Art

[0002] In the field of plant tissue culture, the rapid propagation and efficient planting of medicinal plants have always been the focus of research. Traditional plant propagation methods, such as seed propagation and cutting propagation, often have problems such as slow propagation speed, high environmental requirements and low success rate. Tissue culture technology has become an important research direction in the field of modern agriculture and biotechnology due to its advantages such as being able to produce a large number of plants in a short period of time, maintaining genetic stability and not being restricted by seasons.

[0003] The main challenges faced by tissue culture propagation of medicinal plants include: how to improve the differentiation rate and rooting rate, how to shorten the culture cycle, how to ensure the maintenance of sterile conditions during tissue culture, and how to optimize the culture medium composition to promote the healthy growth of plants and the accumulation of medicinal ingredients.

[0004] Although the traditional tissue culture method has made some progress, it still has some shortcomings:

[0005] Low differentiation and rooting efficiency: Many medicinal plants face the problem of low differentiation and rooting rates in tissue culture, which limits the wide application of their tissue culture; long culture cycle: conventional culture conditions often cannot provide the optimal growth environment, resulting in a long culture cycle and affecting the reproduction efficiency; the composition of nutrient solution and culture medium is single: the composition of traditional culture medium is relatively fixed, which makes it difficult to meet the nutritional needs of different plants or the same plant at different development stages; inflexible light environment control: lighting conditions are usually fixed and cannot be adjusted according to different stages of plant development, affecting the accumulation of medicinal ingredients; high risk of contamination: the difficulty of aseptic operation and improper operation may lead to contamination, reducing the success rate of tissue culture.

[0006] The above technical background reveals the challenges faced by the rapid propagation technology of medicinal plant tissue culture, as well as the need for more efficient and flexible culture methods. New technical methods need to integrate intelligent control technology, optimize culture medium formula and dynamic light regulation means to improve tissue culture efficiency and yield. This provides a background basis for the present invention and illustrates the necessity of improvement and innovation on the existing technology. Summary of the invention

[0007] 1. Technical problems to be solved

[0008] In view of the problems existing in the prior art, the purpose of the present invention is to provide a method for rapid propagation of medicinal plants by tissue culture, which can achieve real-time adjustment of immersion frequency and time, and add a dynamic circulation stirring function to ensure the uniformity and long-term effectiveness of the nutrient solution, thereby improving the growth efficiency of tissue culture seedlings.

[0009] 2. Technical solution

[0010] To solve the above problems, the present invention adopts the following technical solutions.

[0011] A method for rapid propagation of medicinal plants through tissue culture, comprising the following steps:

[0012] 1) Efficient plant selection and cultivation: Select excellent mother plants with significant medicinal activity and strong growth ability, extract their stem segments or leaf tissues using aseptic operation, inoculate them on a differentiation medium containing induction factors and active excipients, and increase the differentiation rate by regulating the oxygen content and light intensity in the initial culture environment;

[0013] 2) Intelligent intermittent immersion culture: Use an intelligently controlled intermittent immersion system to transfer tissue culture seedlings into liquid culture medium. Use a control device to adjust the immersion frequency and time in real time. The immersion frequency is 6±1 times per day, each time is 8-10 minutes, the culture temperature is 26-28°C, and a dynamic circulation stirring function is added to maintain the uniformity of the nutrient solution;

[0014] 3) Aseptic transplanting and rapid propagation: When the culture reaches the optimal transplanting period, it is transferred through a double-filtered sterile environment to an optimized propagation medium. The propagation efficiency is further improved by precisely controlling the light cycle and temperature conditions.

[0015] Furthermore, in step 2), the ingredients of the nutrient solution are configured in the following proportions (by weight): 2.0±0.5 parts of ammonium nitrate, 5.5±0.5 parts of agar, 11±1 parts of soybean oligosaccharides, 5±1 parts of sodium chloride, 1±0.5 parts of indolebutyric acid, 0.2±0.1 parts of L-serine, and 25±5 parts of water.

[0016] Furthermore, the pH of the nutrient solution was adjusted to pH 6.5±0.5, and ultrasound-assisted sterilization was used to reduce decomposition of the culture solution and extend the use period.

[0017] Furthermore, the filling machine in the intermittent immersion device has a rotation speed of 320±10 r / min, a stirring time of 22±3 minutes, and the deionized water used accounts for 42±3% of the total volume of the nutrient solution.

[0018] Furthermore, during the intermittent immersion culture process, a liquid culture medium treated with microfiltration was added, and the composition of the liquid culture medium was: MS+1.0±0.2 mg / L 6-BA+0.02±0.01 mg / L NAA+30±5 g / L sucrose.

[0019] Furthermore, the medicinal plants include but are not limited to Astragalus, Platycodon and Pinellia, and those with higher contents of medicinal active ingredients are preferably selected.

[0020] Furthermore, the propagation medium is prepared according to the following formula (by weight): 8±2 parts of peeled banana, 2±1 parts of activated carbon, 0.7±0.1 parts of agar, 2.5±0.5 parts of Tween 80, 1.5±0.5 parts of brassinolide, 0.9±0.2 parts of calcium chloride, and 0.6±0.2 parts of soybean oligosaccharides.

[0021] Furthermore, by introducing multi-stage spectral irradiation technology, the ratio of red and blue light is adjusted according to the different developmental stages of medicinal plants during the cultivation process to optimize the growth rate and accumulation of medicinal ingredients.

[0022] 3. Beneficial effects

[0023] Compared with the prior art, the advantages of the present invention are:

[0024] 1) The present invention significantly improves the differentiation rate and rooting rate of plants through optimized culture medium components and intelligent intermittent immersion system. For example, in the experiment of Astragalus, the differentiation rate and rooting rate increased by 25% and 29% respectively; in the experiment of Pinellia, these two indicators increased by 22% and 26% respectively.

[0025] 2) This method greatly shortens the culture cycle and improves tissue culture efficiency. In the tissue culture of Astragalus and Pinellia, the culture cycle was shortened by 38% and 37% respectively, making rapid propagation possible. By fine-tuning the culture conditions, such as photoperiod, temperature, and the composition of the nutrient solution, the fresh weight of individual plants was significantly increased. For example, the fresh weight of individual Astragalus plants increased by 50%, while that of Pinellia increased by 55%.

[0026] 3) By adjusting the ingredients of the nutrient solution and culture medium, such as adding soybean oligosaccharides, brassinolide and peeled bananas, a variety of nutrients and growth factors required for plant growth are provided, promoting the health and rapid growth of plants. Using intelligent control technology, the immersion frequency and time are adjusted in real time, and a dynamic circulation stirring function is added to ensure the uniformity and long-term effectiveness of the nutrient solution, thereby improving the growth efficiency of tissue culture seedlings.

[0027] 4) Transplanting and cultivation in a sterile environment reduces the risk of contamination and improves the success rate of the entire tissue culture process. By adjusting the ratio of red and blue light at different development stages, the growth rate of medicinal plants and the accumulation of medicinal ingredients are promoted, further improving the quality and yield of the plants. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present invention will be described clearly and completely below; obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments, and all other embodiments obtained by ordinary technicians in this field without making creative work based on the embodiments of the present invention are within the scope of protection of the present invention.

[0029] Example 1: Rapid propagation method based on tissue culture of Astragalus

[0030] Experimental steps:

[0031] 1) Plant selection and cultivation: Select excellent mother plants of Astragalus, extract healthy stem tissue from them, and inoculate them in an optimized differentiation induction medium (MS medium supplemented with 1.0 mg / L 6-BA, 0.02 mg / L NAA and 30 g / L sucrose, pH 6.5), and add 0.5% activated carbon to the medium to promote differentiation. The culture conditions are 26°C, light intensity 2000 lux, and photoperiod of 16 hours light / 8 hours dark.

[0032] 2) Intermittent immersion culture: The differentiated tissue culture seedlings were transferred into an intelligent intermittent immersion system. The liquid culture medium consisted of MS + 1.0 mg / L 6-BA + 0.02 mg / L NAA + 30 g / L sucrose. The immersion frequency was 6 times a day, 8 minutes each time. The culture temperature was 27°C and the pH was 6.5.

[0033] 3) Transplantation and propagation: After 25 days of cultivation, transfer to propagation medium (8 parts of peeled banana, 2 parts of activated carbon, 0.7 parts of agar, 1.5 parts of brassinolide, 0.9 parts of calcium chloride, and 0.6 parts of soybean oligosaccharides) and continue to cultivate for 30 days for propagation.

[0034] Experimental data:

[0035]

[0036]

[0037] Result analysis:

[0038] Example 1 significantly improves the tissue culture efficiency of Astragalus membranaceus, shortens the culture period, and improves the differentiation rate, rooting rate, and fresh weight of a single plant by optimizing the culture medium composition and the intelligent intermittent immersion system.

[0039] Example 2: Rapid propagation method based on tissue culture of Pinellia ternata

[0040] Experimental steps:

[0041] 1) Plant selection and cultivation: Select excellent mother plants of Pinellia ternata, extract healthy parts from their leaf tissues, and inoculate them into a solid differentiation medium containing peeled bananas (10 parts) and Tween 80 (2.5 parts). The pH of the medium is adjusted to 6.5, the photoperiod is 14 hours light / 10 hours dark, and the culture temperature is 28°C.

[0042] 2) Intermittent immersion culture: The differentiated seedlings were transferred to liquid culture medium (2.0 parts of ammonium nitrate, 5 parts of sodium chloride, 11 parts of soybean oligosaccharides, 5.5 parts of agar, and 25 parts of water) and cultured using an intelligent intermittent immersion system. The immersion frequency was 7 times a day, each time for 9 minutes. The liquid culture medium was sterilized using ultrasound-assisted technology.

[0043] 3) Transplantation and propagation: Transplant the well-growing tissue culture seedlings to a propagation medium containing brassinolide and calcium chloride, and continue culturing for 35 days to obtain a large number of tissue culture seedlings.

[0044] Experimental data:

[0045] project Traditional methods Example 1 Increase Differentiation rate (%) 72 88 +22％ Rooting rate (%) 65 82 +26％ Fresh weight per plant (g) 0.9 1.4 +55％ Cultivation period (days) 95 60 -37％

[0046] Result analysis:

[0047] Example 2 not only improves the tissue culture efficiency but also significantly shortens the reproduction cycle by optimizing the culture medium for Pinellia ternata and applying the intelligent intermittent immersion technology, demonstrating excellent technical effects.

[0048] The above is only a preferred specific implementation of the present invention; however, the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solution and its improved conception within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for rapid propagation of medicinal plants through tissue culture, characterized in that The steps include: 1) Efficient plant selection and cultivation: Select excellent mother plants with significant medicinal activity and strong growth ability, extract their stem segments or leaf tissues using aseptic operation, inoculate them on a differentiation medium containing induction factors and active excipients, and increase the differentiation rate by regulating the oxygen content and light intensity in the initial culture environment; 2) Intelligent intermittent immersion culture: Using an intelligently controlled intermittent immersion system, the tissue culture seedlings are transferred to the liquid culture medium. The control device is used to adjust the immersion frequency and time in real time. The immersion frequency is 6±1 times per day, each time is 8-10 minutes, the culture temperature is 26-28°C, and a dynamic circulation stirring function is added to maintain the uniformity of the nutrient solution; 3) Aseptic transplanting and rapid propagation: When the culture reaches the optimal transplanting period, it is transferred through a double-filtered sterile environment to an optimized propagation medium. The propagation efficiency is further improved by precisely controlling the light cycle and temperature conditions.

2. A method for rapid propagation of medicinal plants through tissue culture according to claim 1, characterized in that: In the step 2), the ingredients of the nutrient solution are configured in the following proportions (by weight): 2.0±0.5 parts of ammonium nitrate, 5.5±0.5 parts of agar, 11±1 parts of soybean oligosaccharides, 5±1 parts of sodium chloride, 1±0.5 parts of indolebutyric acid, 0.2±0.1 parts of L-serine, and 25±5 parts of water.

3. A method for rapid propagation of medicinal plants through tissue culture according to claim 1, characterized in that: The pH of the nutrient solution was adjusted to pH 6.5 ± 0.5, and ultrasound-assisted sterilization was used to reduce decomposition of the culture solution and extend the use period.

4. A method for rapid propagation of medicinal plants through tissue culture according to claim 1, characterized in that: The filling machine in the intermittent immersion device has a rotation speed of 320±10 r / min, a stirring time of 22±3 minutes, and the deionized water used accounts for 42±3% of the total volume of the nutrient solution.

5. A method for rapid propagation of medicinal plants through tissue culture according to claim 1, characterized in that: During the intermittent immersion culture process, a liquid culture medium treated with microfiltration was added, and the composition of the liquid culture medium was: MS+1.0±0.2mg / L 6-BA+0.02±0.01mg / L NAA+30±5g / L sucrose.

6. A method for rapid propagation of medicinal plants through tissue culture according to claim 1, characterized in that: The medicinal plants include but are not limited to Astragalus, Platycodon and Pinellia, and individuals with higher content of medicinal active ingredients are preferably selected.

7. A method for rapid propagation of medicinal plants through tissue culture according to claim 1, characterized in that: The enrichment medium is prepared according to the following formula (by weight): 8±2 parts of peeled banana, 2±1 parts of activated carbon, 0.7±0.1 parts of agar, 2.5±0.5 parts of Tween 80, 1.5±0.5 parts of brassinolide, 0.9±0.2 parts of calcium chloride, and 0.6±0.2 parts of soybean oligosaccharides.

8. A method for rapid propagation of medicinal plants through tissue culture according to claim 1, characterized in that: By introducing multi-stage spectral irradiation technology, the ratio of red and blue light is adjusted according to the different developmental stages of medicinal plants during the cultivation process to optimize the growth rate and accumulation of medicinal ingredients.

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