A kind of efficient propagation method of Codonopsis pilosula

Abstract

The invention discloses a kind of efficient propagation method of Codonopsis pilosula, by using culture medium A in bottle to complete callus induction, then using culture medium B to complete the induction and proliferation steps of adventitious buds, the proliferation coefficient reaches 45.0-50.0;Then cooperate with "micro-cutting" outside the bottle, so that the rooting rate of adventitious buds reaches more than 90%, and subsequent domestication and transplanting steps can be smoothly carried out, and then after continuing to grow and grow, the rooting seedlings with thick roots and strong seedlings are obtained, and the seedling hardening and transplanting are carried out, and the survival rate after transplanting is also more than 90%. The present invention not only simplifies the tissue culture production process of Codonopsis pilosula seedlings, and the proliferation coefficient is much higher than the existing reports of other species of the same family, and the efficient "micro-cutting" rooting system combines adventitious root induction and domestication, greatly shortens the seedling raising cycle of Codonopsis pilosula tissue culture seedlings, solves the low proliferation coefficient in the tissue culture process of Codonopsis pilosula plants in Campanulaceae, the phenomenon such as difficult rooting and domestication, and low cost, short cycle, high quality and survival rate.

Description

Technical Field

[0001] The invention relates to the technical field of medicinal plant cultivation, and in particular to a high-efficiency propagation method for Codonopsis pilosula. Background Art

[0002] The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.

[0003] Codonopsis pilosula (Franch.) Nannf. is a perennial herbaceous vine of the genus Codonopsis in the family Campanulaceae. It grows in mountain forests and shrubs at an altitude of 1560-3100m. Codonopsis has been used as food and folk medicine in Asian countries such as China, Japan and South Korea for thousands of years. The dried roots of Codonopsis pilosula, C.pilosula Nannf.var.modesta (Nannf.)LTShen and C.tangshen Oliv are included in the Chinese Pharmacopoeia and together constitute the original plants of the Chinese medicine Codonopsis. Traditional Chinese medicine believes that the dried roots of these three plants have the effects of strengthening the spleen and lungs, nourishing blood and promoting the production of body fluids, and can treat spleen and lung qi deficiency, cough, asthma, qi and blood deficiency and other diseases. The various secondary metabolites in the roots of Codonopsis pilosula, such as saponins, polysaccharides, triterpenes, alkaloids, etc., make Codonopsis pilosula have a variety of pharmacological activities, including immunomodulatory, antioxidant, antiviral, anticancer, anti-inflammatory and other functions. Unique biological activity and non-toxicity make Codonopsis pilosula popular for medicinal and edible use. Because Codonopsis pilosula has similar biological activity to ginseng and is cheap, it is often used as a substitute for Panax ginseng C.A.Mey. The Chinese Pharmacopoeia records more than 100 traditional Chinese medicine preparations containing Codonopsis pilosula roots or root extracts. At the same time, Codonopsis pilosula also contains nutrients such as protein, vitamins, and minerals, and often appears in the form of health products among the people, especially for making soup, tea, porridge, and wine. In addition, a large number of chemical components in the above-ground part of Codonopsis pilosula are the same as those in the underground part, which can be processed into feed for sale, and the antioxidant activity, protein, amino acids, total flavonoids, and total polyphenols in the leaves are higher than those in the underground part. This medicinal plant with commercial value in its entirety gives Codonopsis pilosula a huge market potential.

[0004] The history of the use of Codonopsis pilosula has been recorded in various parts of my country. Its wide range of uses has greatly increased the demand for Codonopsis pilosula in the domestic and foreign markets. Uncontrolled excavation has also reduced the natural population of Codonopsis pilosula. There is an urgent need to study the artificial propagation of this important medicinal plant. Codonopsis pilosula is cultivated in various parts of China, and seed propagation is still the main propagation method. Cross-species free pollination, disease transmission and changes in the cultivation environment can easily lead to low quality and low yield consequences of traditional seed propagation of Codonopsis pilosula. At the same time, Codonopsis pilosula seeds are extremely small and easily mutated. The germinated seedlings are weak and entangled with each other. It takes one year to transplant seedlings, which is time-consuming and labor-intensive. Therefore, a fast and efficient method is needed to obtain a large number of genetically stable seedlings, and tissue culture is a sustainable solution for large-scale production of high-quality Codonopsis pilosula. Tissue culture technology not only has the advantages of fast growth, short cycle and strong repeatability, but also has a single source of materials, which can retain the original excellent traits of the plant and obtain a large number of seedlings with highly consistent genetic properties in a short period of time. The development of plant in vitro culture technology provides an effective guarantee for the scarcity of medicinal plant resources. There are some reports on artificial rapid propagation of Codonopsis pilosula, but they are all of low quality, lack of substantial data, and extremely poor reproducibility at the time of publication, so that the overall application of its tissue culture seedlings is still at a low level. Therefore, it is necessary to seek a new asexual propagation method with strong reproducibility, low cost, short time, high quality and survival rate, and the ability to fix excellent traits to expand the propagation of Codonopsis pilosula, especially high-quality seedlings, and to carry out factory production of high-quality seedlings to meet planting needs. Summary of the invention

[0005] The purpose of the invention is to provide an efficient propagation method for Codonopsis pilosula in view of the problems that the current Codonopsis pilosula seed propagation is prone to variation and the tissue culture technology has poor repeatability, solve the problems of low proliferation coefficient and difficult rooting and domestication in the tissue culture process of Codonopsis pilosula plants of the Campanulaceae family, and has low cost, short cycle, high quality and survival rate.

[0006] The technical solution of the present invention is as follows:

[0007] An efficient propagation method for Codonopsis pilosula comprises the following steps:

[0008] Step (1): tissue culture, including explant acquisition, callus induction, adventitious bud differentiation and proliferation;

[0009] Step (2): micro-cutting; transferring the adventitious buds induced in step (1) into the matrix outside the bottle;

[0010] Step (3): Transplanting.

[0011] According to a preferred embodiment, step (1) specifically includes the following sub-steps:

[0012] Step (1.1): Explant acquisition: Select healthy plants with good growth potential and no pests and diseases, take stem segments with 1-2 nodes, and disinfect the stem segments with nodes;

[0013] Step (1.2): Callus induction: The noded stem segments disinfected in step (1.1) are inoculated into callus induction medium A, and callus induction is performed under conditions of controlled light intensity, temperature, and illumination time.

[0014] Step (1.3): Adventitious bud differentiation and proliferation: Cut the callus tissue in step (1.2) into appropriate sizes and transfer them to adventitious bud induction medium B, and culture them under the conditions of controlled light intensity, temperature and lighting time; repeat the above steps for the cultured adventitious buds: divide the adventitious buds into clusters of 3-5 plants and transfer them with a small amount of callus tissue into fresh medium B for propagation, and determine the number of repetitions based on the required seedling base.

[0015] By adopting this method, the reproduction coefficient can reach above 45.0, which is much higher than the existing related reports of Codonopsis plants in the Campanulaceae family. It can quickly achieve the rooting and mass reproduction of Codonopsis, and obtain rooted Codonopsis seedlings with strong seedlings and thick roots.

[0016] According to a preferred embodiment, step (2) micro-cutting specifically includes the following steps: taking the adventitious buds of step (1.3), cutting each bud into 2-3 cm with 2 nodes, gently inserting them into a high-temperature sterilized matrix, lightly spraying sterile water on the surface of the matrix, controlling the ambient light to be greater than: 1500lx; the ambient temperature is 20-25°C, the humidity is 80-90%, and culturing is 30-40d.

[0017] According to a preferred embodiment, the transplanting step (3) specifically comprises the following steps: transferring to a natural environment for a transition period of 5 days, and lightly spraying 100 mL of tap water every day after sunset. After the transition period, each plant root is attached to the substrate and transplanted into a flower pot filled with humus soil to obtain a planted seedling.

[0018] Furthermore, the method for disinfecting the noded stem segments described in step (1.1) is as follows: rinse with running water for 30 minutes, place on a clean bench, treat with 0.1% mercuric chloride (HgCl2) aqueous solution (w / v) for 6-10 minutes, rinse with sterile water 3 times, each time for not less than 3 minutes; shake the vessel thoroughly during the entire disinfection process, and finally place the material on sterile absorbent paper, absorb the surface moisture, and then use a sterile scalpel to cut off 0.2 cm of the wound on the stem of the material.

[0019] According to a preferred embodiment, the callus induction medium A comprises the following components: 6-benzylaminopurine (6-BA), α-naphthylacetic acid (NAA), sucrose and agar powder.

[0020] Furthermore, the callus induction medium A includes components with the following concentrations: 1.5-2.0 mg / L 6-benzylaminopurine (6-BA), 0.5-1.0 mg / L α-naphthylacetic acid (NAA), 30000 mg / L sucrose and 4700 mg / L agar powder.

[0021] Furthermore, the pH value of the culture medium A is 5.4-5.8.

[0022] According to a preferred embodiment, the adventitious bud induction medium B comprises the following components: 6-benzylaminopurine (6-BA), kinetin (KT), α-naphthylacetic acid (NAA), coconut juice, sucrose and agar powder.

[0023] According to a preferred embodiment, the adventitious bud induction medium B comprises components of the following concentrations: 6-benzylaminopurine (6-BA) 1.0-1.5 mg / L, kinetin (KT) 0.5-1.0 mg / L, α-naphthylacetic acid (NAA) 0.1-0.5 mg / L, coconut juice 50-60 mL / L, sucrose 30000 mg / L and agar powder 4700 mg / L.

[0024] The culture medium can be used for both cluster bud induction and proliferation, and has good effects of inducing bud generation and proliferation, thus simplifying the experimental process.

[0025] 6-benzylaminopurine (6-BA) and α-naphthaleneacetic acid (NAA) in the culture medium synergistically promote the growth of clustered buds; at the same time, the addition of coconut juice greatly promotes the production of clustered buds, and the combined effect promotes the generation and proliferation of clustered buds.

[0026] Furthermore, the pH value of the culture medium B is 5.4-5.8.

[0027] Furthermore, the matrix in step (2) is peat soil: perlite = 3:1, the sterilization temperature is 125°C, and the time is 25 minutes; the matrix thickness is 4-6 cm, and the matrix is ​​soaked with sterile water for use.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. An efficient propagation method for Codonopsis pilosula. The present invention uses tissue culture technology to expand and propagate again in combination with micro-cuttings to promote rooting, thereby overcoming the problem of difficult rooting of Codonopsis pilosula tissue culture. Since the callus has a good proliferation effect and a strong redifferentiation ability, and each stem segment of the regenerated plant can become a transfer material, its proliferation coefficient reaches more than 45.0, achieving efficient and rapid propagation. 30 days is a proliferation culture cycle, which greatly exceeds the propagation coefficient reported for plants of the same genus. While meeting the needs of large-scale production, the excellent traits of the mother plant are maintained, and the quality of seedlings is improved;

[0030] 2. An efficient propagation method for Codonopsis pilosula solves the problem of different seed quality caused by large separation of offspring and unstable traits in traditional seed propagation. Through the present invention, all seedlings can maintain the same genotype background, which is easy to standardize and operate in a factory, effectively improving the quality of seedlings, and providing high-quality seedlings of unified standards for large-scale planting;

[0031] 3. An efficient propagation method for Codonopsis pilosula, the invention organically combines adventitious root induction and domestication, and the implementation of in vitro "micro-cutting" integrates the rejuvenation, rooting and domestication of weak clump seedlings, which not only greatly shortens the seedling raising period, but also solves the problem of high mortality rate of domestication of tissue culture seedlings that has always troubled breeders in Codonopsis pilosula; in vitro "micro-cutting" makes the survival rate of seedlings transplanted high and grows rapidly; currently, demonstration cultivation has been carried out with good results;

[0032] 4. An efficient propagation method for Codonopsis pilosula. In addition to callus induction culture, proliferation and redifferentiation are all carried out in the same culture medium, which simplifies the culture procedure; the entire rapid propagation process only requires one culture medium to complete the cycle of callus-adventitious buds-callus-adventitious buds, which is conducive to arranging production plans;

[0033] 5. An efficient propagation method for Codonopsis pilosula. The present invention uses stem segments with nodes of Codonopsis pilosula as explants. Experiments show that the culture effect is much better than that of stem tips and leaves, and the callus induction rate of stem segments is higher than that of stem tips and leaves. Since the stem segments lose the suppression of apical dominance, the ratio of endogenous auxin to cytokinin is lower than that of stem tips. In addition, trauma accelerates the synthesis of cytokinin at the wound, and higher cytokinin promotes the formation of callus at the wound. It is speculated that this is the reason why the callus induction rate of stem segments is higher than that of stem tips.

[0034] 6. A highly efficient propagation method for Codonopsis pilosula. In addition to achieving highly efficient proliferation of callus tissue-adventitious cluster buds, the present invention also found that Codonopsis pilosula can also produce regenerated buds through a direct route. Base stem cluster buds will appear on the nodes at the base of the stem segment, and the proliferation of the apical buds of the regenerated buds and the formation of axillary buds on the nodes will greatly increase the propagation efficiency of Codonopsis pilosula. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a schematic diagram of the results of callus induction, proliferation and adventitious bud growth in Example 3, scale bar = 1.5 cm;

[0036] in Figure 1 A is the callus image of the noded stem segment 15 days after grafting; Figure 1 B is the picture of rapid growth of callus tissue after 25 days; Figure 1 C and D are 30 days later, the callus will continue to turn green and form a few green bud spots on the surface; Figure 1 E: The emergence of adventitious buds can be clearly seen 5 days after callus transfer; Figure 1 F is a picture showing that callus tissue continues to increase and adventitious buds continue to grow 10 days after callus transfer; Figure 1 G is the picture of adventitious cluster buds after 20 days of cultivation; Figure 1 H means that after 30 days of culture, the adventitious buds grew vigorously and covered the entire culture medium. At this time, the proliferation coefficient was between 45.0 and 50.0; Figure 1 I is the basal stem clustered buds that appear during callus induction; Figure 1 J is the axillary bud formed by adventitious buds of callus tissue;

[0037] Figure 2 This is a schematic diagram of the rooting and transplanting results inside and outside the bottle in Example 3, scale = 1.5 cm.

[0038] in Figure 2 A and B are pictures of the rooting process in the bottle, in which the leaves at the base of the regenerated buds turn yellow and fall off, and some callus tissue appears at the incision; Figure 2 C, D Wait until the test tube seedlings grow to 2-3cm or cut the clustered buds into 2-3cm pieces and insert them into the medium sterilized by high temperature for cultivation; Figure 2 E is the root system of the regenerated plant after 30 days of culture in the foam box; Figure 2 F is a picture of the lush growth of the plant after the seedlings in the substrate were transplanted into the pot and cultured for 30 days. DETAILED DESCRIPTION

[0039] The specific embodiments listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of the present invention. Therefore, the equal transformations and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturers are not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In other embodiments, the methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the purport of the present invention.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. Unless otherwise specified, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0041] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0042] Embodiment 1 A kind of efficient breeding method of Codonopsis pilosula

[0043] Use the following steps:

[0044] Step (1):

[0045] Step (1.1): Obtaining explants: Select healthy plants with good growth potential and no pests and diseases, take stem segments with 1-2 nodes, rinse the stem segments with nodes with running water for 30 minutes, place them on a clean bench, treat them with 0.1% mercuric chloride (HgCl2) aqueous solution (w / v) for 6 minutes, and rinse them with sterile water for 3 times, each time for no less than 3 minutes; shake the container thoroughly during the entire disinfection process, and finally place the material on sterile absorbent paper, absorb the surface moisture, and use a sterile scalpel to cut off 0.2 cm of the wound on the material stem;

[0046] Step (1.2): Callus induction: The noded stem segments disinfected in step (1.1) are vertically inoculated into callus induction medium A in a physiological direction, and callus induction is performed under conditions of controlled light intensity, temperature, and illumination time;

[0047] The formula of medium A is:

[0048] MS basic culture medium:

[0049]

[0050] Culture conditions: Callus induction was carried out under the conditions of 1800-2200lx illumination, 10h / d illumination time, and temperature controlled at 23±2℃. Callus began to appear after 15 days, and a large amount of callus was formed after 20 days; after 30 days, the callus continued to turn green and a few green buds formed on the surface.

[0051] Step (1.3): Adventitious bud differentiation and proliferation: Cut the callus tissue in step (1.2) into appropriate sizes and transfer them to adventitious bud induction medium B, and culture them under the conditions of controlled light intensity, temperature, and lighting time; repeat the above steps for the cultured adventitious buds, divide the adventitious buds into clusters of 3-5 plants and transfer them with a small amount of callus tissue into fresh medium B for propagation, and determine the number of repetitions based on the required seedling base.

[0052] The formula of medium B is:

[0053] MS basic culture medium:

[0054]

[0055] Culture conditions: Culture under the conditions of light intensity of 1500-2000lx, light time of 10h / d, and temperature controlled at 23±2℃; the appearance of adventitious buds can be clearly seen 5 days after transfer; the callus tissue continues to increase 10 days after transfer, and the adventitious buds continue to grow; the adventitious buds gradually increase after 20 days of culture, and the callus tissue at the base also continues to proliferate; after 30 days of culture, the adventitious buds grow vigorously and cover the entire culture medium; at this time, the proliferation coefficient is about 48.60.

[0056] Step (2): Cut each bud into 2-3 cm long with 2 nodes, gently insert them into a foam box filled with a high-temperature sterilized matrix, then lightly spray sterile water on the surface of the matrix, finally seal the foam box with a transparent plastic wrap, and place it in a cool and bright place for cultivation; at this time, the light intensity is about 2000lx, the temperature in the foam box is 20-25°C, and the humidity is about 80-90%. After 30 days, rooted seedlings with strong roots and thick roots can be obtained, and the rooting rate is 93.6%.

[0057] Step (3): After the rooted seedlings in step (2) have been cultured in the foam box for 30 days, the sealing film is opened and the seedlings are placed under natural light for 5 days, during which 100 mL of tap water is lightly sprayed after sunset every day. Then, each seedling is transplanted into a pot filled with humus soil with its root attached to the substrate, thereby obtaining transplanted seedlings with a survival rate of up to 95.5%.

[0058] Embodiment 2 A kind of efficient propagation method of Codonopsis pilosula

[0059] Use the following steps:

[0060] Step (1):

[0061] Step (1.1): Obtaining explants: Select healthy plants with good growth potential and no pests and diseases, take stem segments with 1-2 nodes, rinse the stem segments with nodes with running water for 30 minutes, place them on a clean bench, treat them with 0.1% mercuric chloride (HgCl2) aqueous solution (w / v) for 8 minutes, and rinse them with sterile water for 3 times, each time for no less than 3 minutes; shake the container thoroughly during the entire disinfection process, and finally place the material on sterile absorbent paper, absorb the surface moisture, and use a sterile scalpel to cut off 0.2 cm of the wound on the stem of the material;

[0062] Step (1.2): Callus induction: The noded stem segments disinfected in step (1.1) are vertically inoculated into callus induction medium A in a physiological direction, and callus induction is performed under conditions of controlled light intensity, temperature, and illumination time;

[0063] The formula of medium A is:

[0064] MS basic culture medium:

[0065]

[0066] Culture conditions: Callus induction is carried out under the conditions of light intensity of 1800-2200lx, light time of 10h / d, and temperature controlled at 23±2℃. Callus begins to appear after 15d, and a large amount of callus is formed after 20d. After 30d, the callus will continue to turn green and form a few green buds on the surface.

[0067] Step (1.3): Adventitious bud differentiation and proliferation: Cut the callus tissue in step (1.2) into appropriate sizes and transfer them to adventitious bud induction medium B, and culture them under the conditions of controlled light intensity, temperature, and lighting time; repeat the above steps for the cultured adventitious buds, divide the adventitious buds into clusters of 3-5 plants and transfer them with a small amount of callus tissue into fresh medium B for propagation, and determine the number of repetitions based on the required seedling base.

[0068] The formula of medium B is:

[0069] MS basic culture medium:

[0070]

[0071]

[0072] Culture conditions: Culture under the conditions of light intensity of 1500-2000lx, light time of 10h / d, and temperature controlled at 23±2℃; the appearance of adventitious buds can be clearly seen 5 days after transfer; the callus tissue continues to increase 10 days after transfer, and the adventitious buds continue to grow; the adventitious buds gradually increase after 20 days of culture, and the callus tissue at the base also continues to proliferate; after 30 days of culture, the adventitious buds grow vigorously and cover the entire culture medium; at this time, the proliferation coefficient is about 47.90.

[0073] Step (2): Cut each bud into 2-3 cm long with 2 nodes, gently insert them into a foam box filled with a high-temperature sterilized matrix, then lightly spray sterile water on the surface of the matrix, finally seal the foam box with a transparent plastic wrap, and place it in a cool and bright place for cultivation; at this time, the light intensity is about 2000lx, the temperature in the foam box is 20-25°C, and the humidity is about 80-90%. After 35 days, rooted seedlings with strong roots and thick roots can be obtained, and the rooting rate is 94.5%.

[0074] Step (3): After the rooted seedlings in step (2) are cultured in the foam box for 35 days, the sealing film is opened and the seedlings are placed under natural light for 5 days, during which 100 mL of tap water is lightly sprayed after sunset every day. Then, each seedling is transplanted into a pot filled with humus soil with its root attached to the substrate, thereby obtaining transplanted seedlings with a survival rate of up to 95.8%.

[0075] Embodiment 3 A kind of efficient breeding method of Codonopsis pilosula

[0076] Use the following steps:

[0077] Step (1):

[0078] Step (1.1): Obtaining explants: Select healthy plants with good growth potential and no pests and diseases, take stem segments with 1-2 nodes, rinse the stem segments with nodes with running water for 30 minutes, place them on a clean bench, treat them with 0.1% mercuric chloride (HgCl2) aqueous solution (w / v) for 10 minutes, and rinse them with sterile water for 3 times, each time for no less than 3 minutes; shake the container thoroughly during the entire disinfection process, and finally place the material on sterile absorbent paper, absorb the surface moisture, and use a sterile scalpel to cut off 0.2 cm of the wound on the material stem;

[0079] Step (1.2): Callus induction: The noded stem segments disinfected in step (1.1) are vertically inoculated into callus induction medium A in a physiological direction, and callus induction is performed under conditions of controlled light intensity, temperature, and illumination time;

[0080] The formula of medium A is:

[0081] MS basic culture medium:

[0082]

[0083] Culture conditions: Callus induction was carried out under the conditions of 1800-2200lx illumination, 10h / d illumination time and 23±2℃ temperature. The induction results were as follows: Figure 1 As shown in A and B, callus tissue began to appear after 15 days, and a large amount of callus tissue was formed after 25 days; Figure 1 As shown in C and D, after 30 days, the callus tissue will continue to turn green and form a few green buds on the surface.

[0084] Step (1.3): Adventitious bud differentiation and proliferation: Cut the callus tissue in step (1.2) into appropriate sizes and transfer them to adventitious bud induction medium B, and culture them under the conditions of controlled light intensity, temperature, and lighting time; repeat the above steps for the cultured adventitious buds, divide the adventitious buds into clusters of 3-5 plants and transfer them with a small amount of callus tissue into fresh medium B for propagation, and determine the number of repetitions based on the required seedling base.

[0085] The formula of medium B is:

[0086] MS basic culture medium:

[0087]

[0088]

[0089] Culture conditions: Culture under the conditions of light intensity 1500-2000lx, light duration 10h / d, and temperature controlled at 23±2℃; Figure 1 As shown in E, adventitious buds can be clearly seen 5 days after transfer; Figure 1 As shown in F, 10 days after transfer, the callus tissue continued to increase and the adventitious buds continued to grow; Figure 1 As shown in G, after 20 days of culture, the number of adventitious buds gradually increased, and the callus at the base also continued to proliferate; Figure 1 As shown in H, after 30 days of culture, the adventitious buds grew vigorously and covered the entire culture medium; at this time, the proliferation coefficient was about 48.20. Figure 1 I and Figure 1 As shown in J, Figure 1 I indicates that a large number of basal stem clustered buds appeared during callus induction; Figure 1 J is the axillary bud formed by adventitious buds of callus tissue; the buds of these two parts can be pruned for later micro-cuttings;

[0090] Step (2): Figure 2 As shown in C and D, each bud is cut into 2-3 cm with 2 nodes, and gently inserted into a foam box filled with high-temperature sterilized substrate. Then, sterile water is lightly sprayed on the surface of the substrate. Finally, the foam box is sealed with a transparent plastic wrap and placed in a cool and bright place for cultivation; at this time, the light intensity is about 2000lx, the temperature in the foam box is 20-25℃, and the humidity is about 80-90%. Figure 2 As shown in E, rooted seedlings with strong roots and thick roots can be obtained after 40 days, and the rooting rate is 95%.

[0091] Step (3): After the rooted seedlings in the foam box of step (2) have been cultured for 40 days, the sealing film is opened and the seedlings are placed under natural light for 5 days, during which 100 mL of tap water is lightly sprayed after sunset every day, and then each seedling is transplanted with its root attached to the substrate into a pot filled with humus soil to obtain a transplanted seedling. Figure 2 As shown in F, the survival rate can reach 95.7%.

[0092] Comparative Example 1 Comparison of the effect of rooting of Codonopsis pilosula in a bottle

[0093] The operation of step 1 of this comparative example is the same as that of Example 1. Step (2) is to transfer the clustered buds to the rooting medium for rooting induction. The rooting induction was carried out in more than 20 culture media with different basic culture medium types and exogenous hormones and their mass concentrations. The rooting rate was extremely low. For example, the effect after culturing for 20 days in 1 / 2MS+NAA1.0 mg / L+6-BA0.1 mg / L was as follows: Figure 2 As shown in A and B, Figure 2 A It can be seen that during the rooting process, the leaves at the base of the regenerated buds turned yellow and fell off, and even callus tissue appeared at the wound, and the induced rooting rate was less than 5%.

[0094] Comparative Example 2

[0095] The remaining steps of this embodiment are the same as those of Example 3. The difference from Example 3 is that no coconut juice is added to the culture medium B of this embodiment.

[0096] Experimental results: The callus inoculated into the medium B without coconut juice in this application only produced Figure 1 D shows that the callus tissue is enlarged and cannot produce Figure 1 The large number of clustered shoots shown in E and F make it difficult to induce and proliferate clustered shoots, which is not conducive to the continuation of subsequent processes.

[0097] Comparative Example 3: Adjusting the concentration of dual hormones in culture medium B to verify the experimental effect

[0098] The remaining steps of this embodiment are the same as those of Embodiment 3, except that the concentrations of 6-BA and NAA in culture medium B are adjusted.

[0099] The results showed that when the concentration of 6-BA remained unchanged, the callus induction rate increased first and then decreased with the increase of NAA concentration; when NAA was kept at a low concentration, the callus induction rate increased with the increase of 6-BA.

[0100] High concentration of 6-BA combined with low concentration of NAA can induce a large amount of callus tissue. -1 6-BA+0.5mg·L -1 NAA was the most suitable for inducing callus, and the callus induction rate was as high as 98.76%.

[0101] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. An efficient propagation method for Codonopsis pilosula, characterized in that: The steps include: Step (1): tissue culture, including explant acquisition, callus induction, adventitious bud differentiation and proliferation; the step (1) specifically includes the following sub-steps: Step (1.1): Obtaining explants: Select healthy plants with good growth potential and no pests or diseases, take stem segments with 1-2 nodes, and disinfect the stem segments with nodes; Step (1.2): Callus induction: The noded stem segments disinfected in step (1.1) are inoculated into callus induction medium A, and callus induction is performed under the conditions of controlled light intensity, temperature, and light exposure time; the callus induction medium A comprises: MS basic culture solution, 1.5-2.0 mg / L 6-benzylaminopurine, 0.5-1.0 mg / L α-naphthaleneacetic acid, 30,000 mg / L sucrose, and 4,700 mg / L agar powder; Step (1.3): Adventitious bud differentiation and proliferation: the callus tissue in step (1.2) is cut into appropriate sizes and transferred to adventitious bud induction medium B, and cultured under the conditions of controlled light intensity, temperature and light exposure time; then the adventitious buds are divided into clumps of 3-5 plants and a small amount of callus tissue is transferred to fresh adventitious bud induction medium B for propagation, and the number of propagation is determined according to the required seedling base; the adventitious bud induction medium B comprises: MS basic culture solution, 6-benzylaminopurine 1.0-1.5 mg / L, kinetin 0.5-1.0 mg / L, α-naphthaleneacetic acid 0.1-0.5 mg / L, coconut juice 50-60 mL / L, sucrose 30000 mg / L and agar powder 4700 mg / L; Step (2): Micro-cutting: transfer the adventitious buds induced in step (1) into the matrix outside the bottle; specifically: take the adventitious buds in step (1.3), cut each bud into 2-3 cm with 2 nodes, gently insert it into the matrix sterilized by high temperature, and lightly spray sterile water on the surface of the matrix; Step (3): Transplanting.

2. A highly efficient propagation method for Codonopsis pilosula according to claim 1, characterized in that, The pH value of the adventitious bud induction medium B is 5.4-5.

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3. A highly efficient propagation method for Codonopsis pilosula according to claim 1, characterized in that, After the micro-cuttings in step (2) are completed, the ambient light is controlled to be greater than 1500 lx; ​​the ambient temperature is 20-25°C, the humidity is 80-90%, and the culture is carried out for 30-40 days.

4. A highly efficient propagation method for Codonopsis pilosula according to claim 1, characterized in that, The method for disinfecting the noded stem segment in step (1.1) is as follows: rinse with running water for 30 minutes, place on a clean bench, treat with 0.1% mercuric chloride aqueous solution (w / v) for 6-10 minutes, rinse with sterile water 3 times, each time for not less than 3 minutes; shake the container thoroughly during the entire disinfection process, and finally place the material on sterile absorbent paper, absorb the surface moisture, and then use a sterile scalpel to cut off 0.2 cm of the wound on the stem of the material.

5. A highly efficient propagation method for Codonopsis pilosula according to claim 1, characterized in that, The pH value of the callus induction medium A is 5.4-5.

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6. A highly efficient propagation method for Codonopsis pilosula according to claim 1, characterized in that, The transplanting step (3) specifically comprises the following steps: transferring the rooted seedlings of step (2) to a natural environment for a transition period of 5 days, lightly spraying 100 mL of tap water every day after sunset, and after the transition period, transplanting each plant with its root attached to the substrate into a flower pot filled with humus soil to obtain planted seedlings.

7. A highly efficient propagation method for Codonopsis pilosula according to claim 1, characterized in that, The matrix in step (2) is peat soil:perlite=3:1, the sterilization temperature is 125°C, the time is 25 min; the matrix thickness is 4-6 cm, and the matrix is ​​soaked with sterile water for later use.

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