Rapid propagation method for tissue culture of rhizoma smilacis glabrae

Abstract

The invention discloses a rhizoma smilacis glabrae tissue culture and rapid propagation method which comprises the following steps: S1, washing an induction material with running water, disinfecting, absorbing moisture on the surface layer of the material, then putting the material on a primary induction culture medium for culture, and inducing adventitious buds; s2, performing multiplication culture on the adventitious buds on a multiplication culture medium; inducing cluster buds; s3, putting the cluster buds into a rooting culture medium, and inducing rooting; s4, selecting test-tube plantlets which grow vigorously and have developed root systems from the rhizoma smilacis glabrae subjected to rooting culture, transferring the test-tube plantlets into a room at normal temperature, releasing a cover for 2 days, opening the cover to enable the rhizoma smilacis glabrae seedlings to be in complete contact with air, watering the plant seedlings in the bottle to keep sufficient water in the bottle during the period, taking out the seedlings from the bottle after 3 days, cleaning a culture medium at the roots, transferring the seedlings into a seedling hardening matrix, and transplanting the seedlings into a seedling hardening medium. Shading culture is performed for 30 days, and then conventional cultivation management is performed. The invention establishes a more efficient glabrous greenbrier rhizome tissue culture rapid propagation system, and has a wide market application prospect.

Description

Technical Field

[0001] The invention belongs to the field of Smilax glabra cultivation, and more specifically, relates to a method for rapid propagation of Smilax glabra tissue culture. Background Art

[0002] Smilax glabra is a Chinese medicinal material included in the Pharmacopoeia of the People's Republic of China (Volume 1) (2020 edition). It is the dried rhizome of Smilax glabra Roxb., a plant of the genus Smilax in the family Liliaceae. It is a traditional Chinese medicine commonly used in my country. It has the effects of regulating the middle and stopping diarrhea, strengthening the bones and muscles, and strengthening the spleen and stomach. Its rhizome is rich in starch. In recent years, it has been often used as a health-preserving material to make cakes, brew wine, and make soup. At present, most of the medicinal materials of Smilax glabra come from wild resources. With the increasing attention of society to traditional Chinese medicine and the increasing demand for health and wellness, Smilax glabra has begun to be planted on a large scale, and the scale of seedling cultivation is also expanding. Smilax glabra mainly relies on seed propagation or tuber propagation, but its seed germination rate is low and the uneven germination seriously affects the seed propagation of Smilax glabra. Therefore, through tissue culture technology, a more efficient tissue culture rapid propagation system of Smilax glabra has been established, which has broad market application prospects. Summary of the invention

[0003] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0004] An object of the present invention is to provide a method for rapid propagation of Smilax glabra tissue culture, which can improve the success rate of rapid propagation of Smilax glabra tissue culture, is conducive to efficiently obtaining a large number of Smilax glabra sterile seedlings, and has broad market application prospects.

[0005] In order to achieve these purposes and other advantages of the present invention, a method for rapid propagation of Smilax glabra tissue culture is provided, which comprises the following steps: S1: Explant selection, sterilization and primary culture Rinse the induction material with running water, dry the surface water of the material after disinfection, and then place it on the primary induction medium for culture to induce adventitious buds; Wherein, the induction material is the stem tip or leaf of Smilax glabra; The shoot tip induction medium was MS medium containing 0.5 mg / L 6-BA and 0.2 mg / L IAA; The leaf induction medium was MS medium containing 1.5 mg / L 6-BA, 0.2 mg / L KT, and 0.2 mg / L NAA; S2: Proliferation culture Proliferate the adventitious buds on a proliferation medium to induce clustered buds; Wherein, the proliferation medium is MS medium containing 0.6 mg / L 6-BA, 0.2 mg / L IBA and 0.2 mg / L IAA; S3: Strong seedlings and rooting The clustered shoots are placed in a rooting medium to induce rooting; Among them, the rooting medium is 1 / 2 MS medium containing 0.5 mg / L IBA, 0.3 mg / L NAA and 0.5 g / L activated carbon; S4: Hardening and transplanting After rooting culture, select the test tube seedlings with vigorous growth and well-developed root system and move them into a normal temperature chamber. Loosen the lid for 2 days and then open it to allow the Smilax glabra seedlings to be fully exposed to the air. During this period, water the seedlings in the bottle to keep the bottle sufficient with water. After 3 days, take out the seedlings from the bottle, wash the culture medium at the roots, move them into the seedling hardening medium, and culture them in the shade for 30 days. Then carry out conventional cultivation management.

[0006] Preferably, the method for disinfecting the inducing material is as follows: sterilize with 75% alcohol for 30 seconds, rinse with sterile water for 2-3 times, place the cleaned stem tips / leaves in 0.1% HgCl2 solution, soak and disinfect the stem tips for 7-8 minutes, soak and disinfect the leaves for 6 minutes, rinse with sterile water for 3 times, each time for 5 minutes, and use sterile paper to absorb the moisture on the surface of the experimental material.

[0007] Preferably, the shoot tip induction culture is cultured under a light intensity of 2000 lx, a light duration of 12 h / d, and a temperature of 22-26°C.

[0008] Preferably, the leaf induction culture is cultured under dark conditions at a temperature of 22-26°C.

[0009] Preferably, the seedling hardening medium is peat soil and vermiculite in a volume ratio of 3:1.

[0010] Preferably, the rooting medium further comprises willow bark powder, and the concentration of the willow bark powder is 5-10 g / L.

[0011] Preferably, the willow bark powder is loaded with a magnetic material.

[0012] Preferably, the method for preparing the willow bark powder-loaded magnetic material comprises the following steps: 1) Dry and crush the willow bark, pass it through a 100-mesh sieve, and take the sieved material to obtain willow bark powder; 2) Add distilled water to FeSO4▪7H2O and FeCl3▪6H2O to dissolve, stir while filling with nitrogen, heat to 80°C, continuously add ammonia water to pH 8.0, then add willow bark powder, stir continuously at 80°C for 1 h, cool to room temperature, wash repeatedly with pure water and ethanol, vacuum dry, grind, and pass through a 100-mesh sieve to obtain willow bark loaded with magnetic material.

[0013] Preferably, the mass ratio of FeSO4▪7H2O, FeCl3▪6H2O and willow bark powder is 5:7:5.

[0014] Preferably, the mass concentration of aqueous ammonia is 25%.

[0015] Preferably, in the middle and late stages of root induction, a vertical downward magnetic field is applied to drive the willow bark powder loaded with magnetic material in the rooting medium to move downward.

[0016] The present invention at least includes the following beneficial effects: first, the present invention establishes a more efficient tissue culture rapid propagation system of Rhizoma Smilacis Glabrae, which can improve the success rate of tissue culture rapid propagation of Rhizoma Smilacis Glabrae, is conducive to efficiently obtaining a large number of Rhizoma Smilacis Glabrae sterile seedlings, and has a broad market application prospect. Second, the present invention can promote the growth of the root system by adding willow bark loaded with magnetic material in the rooting medium, so that the root system of the cultivated tissue culture seedling is more developed. Through the synergistic effect of the willow bark loaded with magnetic material and the magnetic field, the growth of the root system is greatly promoted, making the root system more abundant, thereby helping to improve the survival rate of the seedlings.

[0017] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. DETAILED DESCRIPTION

[0018] The present invention is described in further detail below so that those skilled in the art can implement it according to the description.

[0019] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0020] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0021] <Example 1> A method for rapid propagation of Smilax glabra tissue culture, comprising the following steps: S1: Explant selection, sterilization and primary culture Rinse the induction material with running water, dry the surface water of the material after disinfection, and then place it on the primary induction medium for culture to induce adventitious buds; Wherein, the induction material is the leaves of Smilax glabra. The leaf induction medium was MS medium containing 1.5 mg / L 6-BA, 0.2 mg / L KT (kinetin), 0.2 mg / L NAA (naphthaleneacetic acid), and 5 g / L agar; The disinfection method of the induction material is as follows: sterilize with 75% alcohol for 30 seconds, then rinse with sterile water, place the cleaned stem tip / leaf in 0.1% HgCl2 solution, soak the stem tip for 7 minutes, soak the leaf for 6 minutes, rinse with sterile water 3 times, each time for 5 minutes, and use sterile paper to absorb the moisture on the surface of the experimental material; The shoot tip induction culture was cultured under a light intensity of 2000 lx, a light duration of 12 h / d, and a temperature of 22-26°C; Leaf induction culture was cultured in dark conditions at 22-26 °C; S2: Proliferation culture Proliferate the adventitious buds on a proliferation medium to induce clustered buds; Among them, the proliferation medium is MS medium containing 0.6 mg / L 6-BA, 0.2 mg / L IBA (indole-3-butyric acid), 0.2 mg / L IAA, and 5 g / L agar; S3: Strong seedlings and rooting The clustered shoots are placed in a rooting medium to induce rooting; Among them, the rooting medium is 1 / 2 MS medium containing 0.5 mg / L IBA, 0.3 mg / L NAA, 0.5 g / L activated carbon and 5 g / L agar; S4: Hardening and transplanting After the rooting culture, select the test tube seedlings with vigorous growth and well-developed root system and move them into the normal temperature room. After loosening the lid for 2 days, open the lid to let the seedlings of Smilax glabra fully contact with the air. During this period, sprinkle water on the plant seedlings in the bottle to keep the water in the bottle sufficient. After 3 days, take out the seedlings from the bottle, wash the culture medium on the roots, move them into the seedling hardening medium, shade and culture for 30 days, and then carry out the conventional cultivation management. The seedling hardening medium is peat soil and vermiculite in a mass ratio of 3:1.

[0022] <Example 2> A method for rapid propagation of Smilax glabra tissue culture, comprising the following steps: S1: Explant selection, sterilization and primary culture Rinse the induction material with running water, dry the surface water of the material after disinfection, and then place it on the primary induction medium for culture to induce adventitious buds; Among them, the induction material is the stem tip of Smilax glabra. The shoot tip induction medium was MS medium containing 0.5 mg / L 6-BA (cytokinin), 0.2 mg / L IAA (indole-3-acetic acid), and 5 g / L agar; The disinfection method of the induction material is as follows: sterilize with 75% alcohol for 30 seconds, then rinse with sterile water, place the cleaned stem tip / leaf in 0.1% HgCl2 solution, soak the stem tip for 7 minutes, soak the leaf for 6 minutes, rinse with sterile water 3 times, each time for 5 minutes, and use sterile paper to absorb the moisture on the surface of the experimental material; The shoot tip induction culture was cultured under a light intensity of 2000 lx, a light duration of 12 h / d, and a temperature of 22-26°C; Leaf induction culture was cultured in dark conditions at 22-26 °C; S2: Proliferation culture Proliferate the adventitious buds on a proliferation medium to induce clustered buds; Among them, the proliferation medium is MS medium containing 0.6 mg / L 6-BA, 0.2 mg / L IBA (indole-3-butyric acid), 0.2 mg / L IAA, and 5 g / L agar; S3: Strong seedlings and rooting The clustered shoots are placed in a rooting medium to induce rooting; Among them, the rooting medium is 1 / 2 MS medium containing 0.5 mg / L IBA, 0.3 mg / L NAA, 0.5 g / L activated carbon and 5 g / L agar; S4: Hardening and transplanting After the rooting culture, select the test tube seedlings with vigorous growth and well-developed root system and move them into the normal temperature room. After loosening the lid for 2 days, open the lid to let the seedlings of Smilax glabra fully contact with the air. During this period, sprinkle water on the plant seedlings in the bottle to keep the water in the bottle sufficient. After 3 days, take out the seedlings from the bottle, wash the culture medium on the roots, move them into the seedling hardening medium, shade and culture for 30 days, and then carry out the conventional cultivation management. The seedling hardening medium is peat soil and vermiculite in a volume ratio of 3:1.

[0023] <Example 3> A method for rapid propagation of Smilax glabra tissue culture, comprising the following steps: S1: Explant selection, sterilization and primary culture Rinse the induction material with running water, dry the surface water of the material after disinfection, and then place it on the primary induction medium for culture to induce adventitious buds; Among them, the induction material is the stem tip of Smilax glabra. The shoot tip induction medium was MS medium containing 0.5 mg / L 6-BA, 0.2 mg / L IAA, and 5 g / L agar; The method of disinfecting the induction material is as follows: sterilize with 75% alcohol for 30 seconds, then rinse with sterile water, place the cleaned stem tip / leaf in 0.1% HgCl2 solution, soak the stem tip for 7-8 minutes, soak the leaf for 6 minutes, rinse with sterile water twice, each time for 5 minutes, and use sterile paper to absorb the moisture on the surface of the experimental material; The shoot tip induction culture was cultured under a light intensity of 2000 lx, a light duration of 12 h / d, and a temperature of 22-26°C; Leaf induction culture was cultured in dark conditions at 22-26 °C; S2: Proliferation culture Proliferate the adventitious buds on a proliferation medium to induce clustered buds; The proliferation medium is MS medium containing 0.6 mg / L 6-BA, 0.2 mg / L IBA, 0.2 mg / L IAA and 5 g / L agar; S3: Strong seedlings and rooting The clustered shoots were placed in rooting medium and cultured for 30 days to induce rooting; The rooting medium was 1 / 2 MS medium containing 0.5 mg / L IBA, 0.3 mg / L NAA, 0.5 g / L activated carbon, 5 g / L willow bark loaded with magnetic materials, and 3 g / L agar; The preparation method of willow bark powder loaded magnetic material comprises the following steps: 1) Dry and crush the willow bark, pass it through a 100-mesh sieve, and take the sieved material to obtain willow bark powder; 2) Add distilled water to FeSO4▪7H2O and FeCl3▪6H2O to dissolve, stir while filling with nitrogen, heat to 80°C, continuously drop ammonia water until the pH is 8.0, then add willow bark powder, stir continuously at 80°C for 1 h, cool to room temperature, wash repeatedly with pure water and ethanol, vacuum dry, grind, and pass through a 100-mesh sieve to obtain willow bark loaded with magnetic material; The mass ratio of FeSO4▪7H2O, FeCl3▪6H2O and willow bark powder is 5:7:5; The mass concentration of ammonia water is 25%; In the middle and late stages of rooting culture, a vertical downward magnetic field was applied to drive the willow bark powder loaded with magnetic materials to move downward. On the 15th day of rooting induction, the powder was first treated with a magnetic field of 20 mT for 0.5 h, and then with a magnetic field of 10 mT until the 18th day. On the 18th day of root induction, the plants were first treated with a 20 mT magnetic field for 1 h, and then with a 10 mT magnetic field until the 21st day. On the 21st day of root induction, the plants were first treated with a magnetic field of 25 mT for 0.5 h, and then with a magnetic field of 10 mT until the 23rd day. On the 23rd day of root induction, the plants were first treated with a 25 mT magnetic field for 1 h, and then with a 10 mT magnetic field until the 25th day. On the 25th day of root induction, the plants were first treated with a 30 mT magnetic field for 0.5 h, and then with a 10 mT magnetic field until the 27th day. On the 27th day of root induction, the plants were first treated with a magnetic field of 35 mT for 0.5 h, and then with a magnetic field of 10 mT until the 29th day. On the 29th day of root induction, the plants were first treated with a 40 mT magnetic field for 0.5 h, and then with a 10 mT magnetic field until the 30th day. S4: Hardening and transplanting After the rooting culture, select the test tube seedlings with vigorous growth and well-developed root system and move them into the normal temperature room. After loosening the lid for 2 days, open the lid to let the seedlings of Smilax glabra fully contact with the air. During this period, sprinkle water on the plant seedlings in the bottle to keep the water in the bottle sufficient. After 3 days, take out the seedlings from the bottle, wash the culture medium on the roots, move them into the seedling hardening medium, shade and culture for 30 days, and then carry out the conventional cultivation management. The seedling hardening medium is peat soil and vermiculite in a volume ratio of 3:1.

[0024] <Effect Test> Comparative Example 1: The method of Example 2 was adopted, except that the proliferation medium was MS medium containing 0.6 mg / L 6-BA, 0.2 mg / L IBA and 5 g / L agar.

[0025] Comparative Example 2: The method of Example 2 was adopted, except that the proliferation medium was MS medium containing 0.6 mg / L 6-BA, 0.2 mg / L IAA and 5 g / L agar.

[0026] Comparative Example 3: The method of Example 3 was adopted, except that a downward magnetic field was applied to the rooting medium to drive the willow bark powder loaded with magnetic material to move downward, and a magnetic field with an intensity of 20 mT was continuously applied from the 15th day to 30 days.

[0027] Comparative Example 4: The method of Example 3 was adopted, except that a downward magnetic field was applied to the rooting medium to drive the willow bark powder loaded with magnetic material to move downward, and a magnetic field with an intensity of 35 mT was continuously applied from the 15th day to 30 days.

[0028] Comparative Example 5: The method of Example 3 was used, except that the 10 mT magnetic field was not continuously used for treatment from d 15 to d 30.

[0029] Comparative Example 6: The method of Example 3 was adopted, except that the willow bark powder was not loaded with magnetic material.

[0030] Comparative Example 7: The method of Example 3 was adopted, except that no magnetic field was applied in the middle and late stages of root induction.

[0031] Comparative Example 8: The method of Example 3 was adopted, except that the rooting medium did not contain willow bark loaded with magnetic material, that is, only magnetic field treatment was applied.

[0032] The numbers of clustered buds of Comparative Example 1, Comparative Example 2 and Example 2 were counted, and the results are shown in Table 1.

[0033] Table 1 Number of clustered buds Quantity (pcs) Comparative Example 2 3 Comparative Example 3 4 Example 2 8 From the results in Table 1, it can be seen that the simultaneous use of IBA and IAA during proliferation culture is conducive to the formation of clustered buds.

[0034] The rooting rate (%) and rooting condition after the rooting induction culture of Example 2 and Example 3 were observed and counted. The results are shown in Table 2. Rooting rate (%) Rooting status Example 2 80.9 Root system is average, without root hairs Example 3 86.8 Well-developed root system with abundant root hairs

[0035] From the results in Table 2, it can be seen that adding willow bark loaded with magnetic materials to the rooting medium can promote the growth of the root system.

[0036] The survival rates after hardening of the seedlings of Example 2, Example 3 and Comparative Examples 2-8, that is, the survival rates after 30 days of hardening and cultivation, are shown in Table 3.

[0037] Table 3. Survival rate of seedling hardening Survival rate (%) Example 2 70 Example 3 94 Comparative Example 3 65 Comparative Example 4 59 Comparative Example 5 83 Comparative Example 6 71 Comparative Example 7 78 Comparative Example 8 72 As can be seen from Table 3, the results of Example 2 and Example 3 show that adding willow bark loaded with magnetic material to the rooting medium and applying a magnetic field with a specific rule can significantly improve the survival rate of the tissue culture seedlings. The results of Example 3, Comparative Example 3, and Comparative Example 4 show that a single high-intensity magnetic field treatment not only fails to improve the survival rate, but also has a negative impact on the survival rate of the tissue culture seedlings; the results of Example 3, Comparative Example 6, and Comparative Example 8 show that only applying a magnetic field or only adding willow bark has little effect on the survival rate of the tissue culture seedlings; the results of Example 2 and Comparative Example 7 show that adding willow bark loaded with magnetic material can improve the survival rate of the tissue culture seedlings to a certain extent. The present invention adds willow bark loaded with magnetic material into the rooting medium, and applies magnetic field treatment with a specific rule during the rooting culture process, which can significantly improve the survival rate of tissue culture seedlings. On the one hand, it may be because the salicylic acid rich in the willow bark cooperates with the iron element to promote the growth of the root system, so that the root system of the tissue culture seedling is more developed, which is beneficial to improving the survival rate of the seedlings. On the other hand, it may be because applying the magnetic field can promote the downward movement of the willow bark loaded with magnetic material, so that the rooting medium becomes more dense, squeezes the root system, makes the root system grow stronger, the root system is more developed, promotes the formation of root hairs, and thus is beneficial to improving the survival rate of the seedlings.

[0038] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the embodiments shown and described herein.

Claims

1. A method for rapid propagation of Smilax glabra tissue culture, characterized in that: The following steps are involved: S1: Explant selection, sterilization and primary culture Rinse the induction material with running water, dry the surface water of the material after disinfection, and then place it on the primary induction medium for culture to induce adventitious buds; Wherein, the induction material is the stem tip or leaf of Smilax glabra; The shoot tip induction medium was MS medium containing 0.5 mg / L 6-BA and 0.2 mg / L IAA; The leaf induction medium was MS medium containing 1.5 mg / L 6-BA, 0.2 mg / L KT, and 0.2 mg / L NAA; S2: Proliferation culture Proliferate the adventitious buds on a proliferation medium to induce clustered buds; Wherein, the proliferation medium is MS medium containing 0.6 mg / L 6-BA, 0.2 mg / L IBA and 0.2 mg / L IAA; S3: Strong seedlings and rooting The clustered shoots are placed in a rooting medium to induce rooting; Among them, the rooting medium is 1 / 2 MS medium containing 0.5 mg / L IBA, 0.3 mg / L NAA and 0.5 g / L activated carbon; S4: Hardening and transplanting After rooting culture, select the test tube seedlings with vigorous growth and well-developed root system and move them into a normal temperature chamber. Loosen the lid for 2 days and then open it to allow the Smilax glabra seedlings to be fully exposed to the air. During this period, water the seedlings in the bottle to keep the bottle sufficient with water. After 3 days, take out the seedlings from the bottle, wash the culture medium at the roots, move them into the seedling hardening medium, and culture them in the shade for 30 days. Then carry out conventional cultivation management.

2. The method for rapid propagation of Rhizoma Smilacis Glabrae through tissue culture according to claim 1, characterized in that: The disinfection method of the induction material is as follows: sterilize with 75% alcohol for 30 seconds, then rinse with sterile water 2-3 times, place the washed stem tips / leaves in 0.1% HgCl2 solution respectively, soak the stem tips for 7-8 minutes, soak the leaves for 6 minutes, rinse with sterile water 3 times, each time for 5 minutes, and use sterile paper to absorb the moisture on the surface of the experimental materials.

3. The method for rapid propagation of Rhizoma Smilacis Glabrae through tissue culture according to claim 1, characterized in that: The shoot tip induction culture was cultured under a light intensity of 2000 lx, a light duration of 12 h / d, and a temperature of 22-26 ℃.

4. The method for rapid propagation of Rhizoma Smilacis Glabrae through tissue culture according to claim 1, characterized in that: Leaf induction culture was cultured in dark conditions at a temperature of 22-26 ℃.

5. The method for rapid propagation of Rhizoma Smilacis Glabrae through tissue culture according to claim 1, characterized in that: The seedling hardening medium is peat soil and vermiculite in a volume ratio of 3:

1.

6. The method for rapid propagation of Rhizoma Smilacis Glabrae through tissue culture according to claim 1, characterized in that: Willow bark powder was also included in the rooting medium at a concentration of 5-10 g / L.

7. The method for rapid propagation of Rhizoma Smilacis Glabrae through tissue culture according to claim 6, characterized in that: Willow bark powder is loaded with magnetic material.

8. The method for rapid propagation of Rhizoma Smilacis Glabrae through tissue culture according to claim 7, characterized in that: The preparation method of willow bark powder loaded magnetic material comprises the following steps: 1) Dry and crush the willow bark, pass it through a 100-mesh sieve, and take the sieved material to obtain willow bark powder; 2) Add distilled water to FeSO4▪7H2O and FeCl3▪6H2O to dissolve, stir while filling with nitrogen, heat to 80°C, continuously add ammonia water to pH 8.0, then add willow bark powder, stir continuously at 80°C for 1 h, cool to room temperature, wash repeatedly with pure water and ethanol, vacuum dry, grind, and pass through a 100-mesh sieve to obtain willow bark loaded with magnetic material.

9. The method for rapid propagation of Rhizoma Smilacis Glabrae through tissue culture according to claim 8, characterized in that: The mass ratio of FeSO4▪7H2O, FeCl3▪6H2O and willow bark powder is 5:7:

5.

10. The method for rapid propagation of Smilax glabra tissue culture according to claim 8, characterized in that: In the middle and late stages of root induction, a vertical downward magnetic field is applied to drive the willow bark powder loaded with magnetic materials in the rooting medium to move downward.