A tissue culture medium and method for rapid propagation of sinomenium acutum

Abstract

The application provides a tissue culture medium for rapid propagation of Caulis Sinomenii and a method for rapid propagation of Caulis Sinomenii, and belongs to the technical field of plant tissue culture.The application provides a tissue culture medium for rapid propagation of Caulis Sinomenii, which comprises an induction medium and a rooting medium; the induction medium takes MS medium as a basic medium and further comprises 1-2 mg / L 6-BA and 0.1-0.2 mg / L NAA; the rooting medium takes 1 / 2MS medium as a basic medium and further comprises 0.1-1 g / L Ac and 0.1-0.2 mg / L NAA.In the embodiment of the application, the stem section with buds of Caulis Sinomenii is used as an explant, the axillary bud differentiation is directly induced by using the tissue culture medium, a large number of seedlings can be obtained, a large number of propagation can be realized in a short time, and the propagation efficiency of Caulis Sinomenii is improved.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to a tissue culture medium for the rapid propagation of *Sinomenium acutum* and a method for the rapid propagation of *Sinomenium acutum*. Background Technology

[0002] *Sinomenium acutum*, a plant of the Menispermaceae family, is used to dispel wind and dampness, clear the meridians, and promote urination. Its main active ingredient is sinomenine. Due to the large amount of sinomenine, a secondary metabolite with high medicinal value, accumulated in its roots, it has attracted increasing attention from scholars and the medical and commercial development sectors both domestically and internationally, and demand is constantly increasing. However, *Sinomenium acutum* is usually harvested by digging it up with its roots, leading to a decline in wild populations. Furthermore, as a perennial woody vine with a long growth period, it is easily affected by factors such as regional differences, climate change, pests and diseases, heavy metals in the soil, and pesticide residues during field cultivation. This results in inconsistent quality of *Sinomenium acutum* sold in the market, leading to poor clinical efficacy in related pharmaceutical preparations and making it difficult to meet the increasingly stringent quality requirements for Chinese medicinal materials in domestic and international markets.

[0003] Plant tissue culture can produce high-quality seedlings that are genetically consistent, disease-free, and vigorous in a short period, ensuring the stability of superior traits and enabling large-scale, year-round production of high-quality plants. Therefore, for *Sinomenium acutum*, utilizing plant tissue culture to expand its propagation and cultivate robust seedlings is of great significance for improving its medicinal quality. However, to date, there are no reports on the biotechnologies related to the rapid propagation of *Sinomenium acutum* through tissue culture. Summary of the Invention

[0004] In view of the deficiencies in the prior art, the purpose of this invention is to provide a tissue culture medium for the rapid propagation of *Sinomenium acutum*, which can achieve rapid propagation of *Sinomenium acutum* using stem segments with axillary buds.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] This invention provides a tissue culture medium for the rapid propagation of *Sinomenium acutum*, including an induction medium and a rooting medium;

[0007] The induction medium is based on MS medium and also includes: 1-2 mg / L 6-BA, 0.1-0.2 mg / L NAA, 6.0-7.0 g / L agar and 30.0-30.5 g / L sucrose;

[0008] The rooting medium is based on 1 / 2 MS medium and also includes: 0.1-1 g / L activated carbon, 0.1-0.2 mg / L NAA, 6.0-7.0 g / L agar and 30.0-30.5 g / L sucrose.

[0009] Preferably, the induction culture medium further includes 0.1–1 g / L activated carbon.

[0010] Preferably, the induction medium is based on MS medium and further includes: 1.5-2 mg / L 6-BA, 0.15-0.2 mg / L NAA, 6.5 g / L agar and 30 g / L sucrose;

[0011] The rooting medium is based on 1 / 2 MS medium and also includes: 0.5-1 g / L activated carbon, 0.15-0.2 mg / L NAA, 6.5 g / L agar, and 30 g / L sucrose.

[0012] This invention provides the application of the tissue culture medium described in the above technical solution in the rapid propagation of *Sinomenium acutum*.

[0013] This invention provides a method for rapid propagation of *Sinomenium acutum*, based on the tissue culture medium described above, comprising:

[0014] Stem segments with axillary buds of *Sinomenium acutum* were induced and cultured in the induction medium to obtain induced seedlings;

[0015] The induced seedlings were cultured in the rooting medium to obtain rooted seedlings.

[0016] Preferably, the induction culture temperature is 24–26°C; and the induction culture time is 28–32 days.

[0017] Preferably, the rooting culture temperature is 24–26°C; and the rooting culture time is 12–30 days.

[0018] Preferably, the stem segment with axillary buds has a length of 0.9–1.2 cm.

[0019] Preferably, before inoculating the stem segments with axillary buds of *Sinomenium acutum* into the induction medium, the process also includes disinfection of the stem segments with axillary buds of *Sinomenium acutum*.

[0020] The disinfection method includes:

[0021] After rinsing the stem segments of *Sinomenium acutum* with axillary buds, soak them in an ethanol aqueous solution and then in a sodium hypochlorite aqueous solution in turn; the soaking time in the sodium hypochlorite aqueous solution is 4 to 7 minutes.

[0022] Preferably, the method further includes: hardening off the rooted seedlings and transplanting them.

[0023] The beneficial effects of this invention are:

[0024] This invention provides a tissue culture medium for the rapid propagation of *Sinomenium acutum*, comprising an induction medium and a rooting medium; the induction medium is based on MS medium and further comprises: 1–2 mg / L 6-BA, 0.1–0.2 mg / L NAA, 6.0–7.0 g / L agar, and 30.0–30.5 g / L sucrose; the rooting medium is based on 1 / 2 MS medium and further comprises: 0.1–1 g / L activated carbon, 0.1–0.2 mg / L NAA, 6.0–7.0 g / L agar, and 30.0–30.5 g / L sucrose. The tissue culture medium provided by this invention contains 6-BA (6-benzylaminopurine), a cytokinin that promotes cell division and expansion, and induces bud formation; NAA (naphthaleneacetic acid), an auxin that promotes the growth and differentiation of axillary buds in *Sinomenium acutum*; the combined action of 6-BA and NAA facilitates the induction of primary cluster buds from *Sinomenium acutum* stem segments with axillary buds, increasing the proliferation coefficient of these cluster buds; Ac (activated carbon) has good adsorption properties and can adsorb harmful substances in the culture medium, including impurities in agar and phenols and quinones secreted by the explants during culture, which are harmful to the explants themselves. 5-hydroxymethylfurfural, produced during autoclaving of sucrose, is beneficial for bud differentiation and proliferation. Adding NAA and Ac to the rooting medium can induce root formation and promote root growth in tissue culture seedlings; the combined action of NAA and Ac can increase the rooting rate. The results of the examples show that using stem segments with buds from *Sinomenium acutum* as explants to directly induce axillary bud differentiation yields a large number of subculture seedlings, enabling mass propagation of seedlings in a short period. Compared to other tissue culture methods, this method eliminates the need for callus culture, reducing steps and improving the propagation efficiency of *Sinomenium acutum*. The tissue culture medium provided by this invention, used for *Sinomenium acutum* tissue culture, features high seedling survival rate, simple operation, short growth cycle, and high technical practicality. In summary, the tissue culture medium provided by this invention, through the combination of components in the induction and rooting media, can significantly improve the survival rate of tissue culture seedlings, achieving the goal of rapid propagation of *Sinomenium acutum*. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 Photograph of a healthy stem segment of *Ipomoea purpurea* with axillary buds;

[0027] Figure 2 A diagram showing the sterilized stem segments of *Sinomenium acutum* with axillary buds inoculated into an induction medium for cultivation.

[0028] Figure 3 Photographs of tissue culture seedlings (induced seedlings) of *Sinomenium acutum* generated through induction culture;

[0029] Figure 4 This is a diagram showing the growth status of explants in the induction medium without added Ac.

[0030] Figure 5 Figure showing the growth status of explants when 0.1 g / L Ac is added to the induction medium;

[0031] Figure 6 This is a diagram showing the growth status of explants when 1 g / L Ac is added to the induction medium.

[0032] Figure 7 Photographs of rooted seedlings after rooting culture;

[0033] Figure 8 This is a diagram showing the growth status of tissue culture seedlings after transplanting. Detailed Implementation

[0034] This invention provides a tissue culture medium for the rapid propagation of *Sinomenium acutum*, including an induction medium and a rooting medium;

[0035] The induction medium is based on MS medium and also includes: 1-2 mg / L 6-BA, 0.1-0.2 mg / L NAA, 6.0-7.0 g / L agar and 30.0-30.5 g / L sucrose;

[0036] The rooting medium is based on 1 / 2 MS medium and also includes: 0.1-1 g / L activated carbon, 0.1-0.2 mg / L NAA, 6.0-7.0 g / L agar and 30.0-30.5 g / L sucrose.

[0037] In the following technical solutions, the present invention does not have any special limitations on the source of each raw material, and any commercially available products in the field can be used.

[0038] In this invention, the tissue culture medium includes an induction medium. In this invention, the induction medium is based on MS medium. In this invention, the amount of MS medium added can be 4.74 g / L. In this invention, the MS medium can meet the nutritional needs of *Sinomenium acutum* axillary buds germinating into tissue culture seedlings, promoting the growth of the induced seedlings.

[0039] In this invention, the induction medium comprises 1–2 mg / L 6-BA, which can be 1.5–2 mg / L, or 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 mg / L. In this invention, the 6-BA in the induction medium promotes cell division and expansion, inducing bud formation.

[0040] In this invention, the induction medium comprises 0.1–0.2 mg / L NAA, which can be 0.15–0.2 mg / L, or 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, or 0.2 mg / L. In this invention, the NAA in the induction medium promotes the growth and differentiation of *Sinomenium acutum* stem segments with axillary buds.

[0041] In this invention, the induction culture medium comprises 6.0–7.0 g / L agar, or 6.5 g / L.

[0042] In this invention, the induction medium comprises 30.0–30.5 g / L sucrose, or 30.0 g / L. In this invention, the 5-hydroxymethylfurfural produced from the sucrose during autoclaving in the induction medium is beneficial for bud differentiation and proliferation.

[0043] In this invention, the induction medium preferably further includes 0.1–1 g / L activated carbon, which can be 0.5–1 g / L, or 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 g / L. In this invention, the activated carbon in the induction medium has good adsorption properties, which can adsorb harmful substances in the medium, including impurities contained in agar, phenols, quinones, and other substances harmful to the explants themselves secreted during culture. This helps reduce the occurrence of explant browning and further improves the emergence rate.

[0044] In this invention, the pH value of the induction culture medium can be 5.8 to 6.0, or it can be 5.8, 5.9 or 6.0.

[0045] The induction culture medium provided by the present invention can induce the growth and differentiation of axillary buds of *Sinomenium acutum*, increase the proliferation coefficient of clustered buds, inhibit explant browning, and thus help improve the emergence rate of *Sinomenium acutum* stem segments with axillary buds.

[0046] In this invention, the tissue culture medium includes a rooting medium. Specifically, the rooting medium is based on 1 / 2 MS medium. The 1 / 2 MS medium is commercially available MS medium at a concentration of 2.37 g / L. This 1 / 2 MS medium meets the nutritional needs of *Sinomenium acutum* tissue culture seedlings during rooting, promoting both seedling growth and root elongation.

[0047] In this invention, the rooting medium includes 0.1–1 g / L activated carbon, which can be 0.5–1 g / L, or 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 g / L. In this invention, the activated carbon in the rooting medium can adsorb harmful substances, reduce browning, and promote root development, thereby increasing the rooting rate.

[0048] In this invention, the rooting medium includes 0.1–0.2 mg / L NAA, which can be 0.15–0.2 mg / L, or 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, or 0.2 mg / L. In this invention, the addition of NAA to the rooting medium can induce the formation of roots in tissue culture seedlings and promote root growth, thus improving the rooting rate.

[0049] In this invention, the rooting medium comprises 6.0–7.0 g / L agar, or 6.5 g / L.

[0050] In this invention, the rooting culture medium comprises 30.0-30.5 g / L sucrose, or it may be 30 g / L.

[0051] In this invention, the pH value of the rooting medium can be 5.8 to 6.0, or it can be 5.8, 5.9 or 6.0.

[0052] The addition of NAA and activated carbon to the rooting culture medium described in the above technical solution of the present invention can promote the root growth of tissue culture seedlings, and the rooting rate can be improved by the combined action of NAA and activated carbon.

[0053] This invention provides the application of the tissue culture medium described in the above-mentioned technical solution in the rapid propagation of *Sinomenium acutum*. The tissue culture medium provided by this invention can induce axillary buds of *Sinomenium acutum* to differentiate into complete plants, improving the emergence rate and rooting rate of tissue-cultured seedlings, thereby achieving rapid propagation of *Sinomenium acutum*. This invention uses budded stem segments of *Sinomenium acutum* as explants and directly induces axillary bud differentiation and rooting using the aforementioned tissue culture medium to obtain a large number of rooted seedlings, enabling mass propagation of seedlings in a relatively short time.

[0054] This invention provides a method for rapid propagation of *Sinomenium acutum*, based on the tissue culture medium described above, comprising:

[0055] Stem segments with axillary buds of *Sinomenium acutum* were induced and cultured in the induction medium to obtain induced seedlings;

[0056] The induced seedlings were cultured in the rooting medium to obtain rooted seedlings.

[0057] This invention involves inducing the growth of *Sinomenium acutum* stem segments with axillary buds in the induction medium to obtain induced seedlings. As an optional embodiment of this invention, the stem segment length can be 0.9–1.2 cm, or 0.9, 1.0, 1.1, or 1.2 cm. Preferably, the *Sinomenium acutum* stem segments with axillary buds include healthy *Sinomenium acutum* stem segments with axillary buds; preferably, each stem segment has one axillary bud. After obtaining the *Sinomenium acutum* stem segments with axillary buds, this invention preferably disinfects them. As an optional embodiment of this invention, the disinfection method includes: rinsing the *Sinomenium acutum* stem segments with axillary buds and then soaking them sequentially in an ethanol aqueous solution and a sodium hypochlorite aqueous solution; the soaking time in the sodium hypochlorite aqueous solution is 4–7 minutes. This invention does not specifically limit the rinsing method; any conventional rinsing method in the art can be used. As an optional embodiment of this invention, the rinsing method can be to rinse with tap water first, and then rinse again with sterile water. After rinsing, the present invention preferably soaks the *Sinomenium acutum* stem segments with axillary buds in an ethanol-water solution; the volume fraction of ethanol in the ethanol-water solution can be 75%; the soaking time can be 30 seconds. After soaking in the ethanol-water solution, the present invention preferably rinses the *Sinomenium acutum* stem segments with axillary buds 3-4 times with sterile water, and then soaks them in a sodium hypochlorite-water solution; the mass fraction of sodium hypochlorite in the sodium hypochlorite-water solution is 2%; the soaking time can be 4-7 minutes, or 4, 5, 6, or 7 minutes, and the present invention preferably shakes the solution continuously during the sodium hypochlorite soaking process. After soaking in the sodium hypochlorite solution, the present invention preferably rinses the *Sinomenium acutum* stem segments with axillary buds 3-4 times with sterile water. After rinsing with sterile water, the obtained *Sinomenium acutum* stem segments with axillary buds are inoculated into an induction culture medium for induction culture.

[0058] As an optional embodiment of the present invention, the induction culture temperature can be 24–26°C or 25°C; the light intensity during the induction culture can be 1000–1500 lx, or 1000, 1100, 1200, 1300, 1400, or 1500 lx; ​​and the induction culture time can be 28–32 days or 30 days. After the induction culture is completed, the present invention yields induced seedlings.

[0059] After obtaining the induced seedlings, the present invention conducts rooting culture on the rooting medium to obtain rooted seedlings. As an optional embodiment of the present invention, the temperature for rooting culture can be 24–26℃, or 25℃; the light intensity for rooting culture can be 1000–1500 lx, or 1000, 1100, 1200, 1300, 1400, or 1500 lx; ​​the rooting culture time can be 12–30 days, or 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. After the rooting culture is completed, the present invention obtains rooted seedlings.

[0060] After obtaining the rooted seedlings, the present invention preferably further includes hardening off and transplanting the rooted seedlings. The present invention does not specifically limit the methods for hardening off and transplanting; conventional hardening off and transplanting methods in the art can be used. As an optional embodiment of the present invention, the hardening off method includes: culturing the rooted seedlings in a rooting medium with a semi-open lid; the semi-open lid culture time can be 5 days. After the semi-open lid culture is completed, the present invention preferably transplants the obtained tissue culture seedlings. The present invention preferably transplants the tissue culture seedlings into a seedling substrate. As an optional embodiment of the present invention, the seedling substrate includes sterilized nutrient soil, peat moss, and vermiculite; the volume ratio of the nutrient soil, peat moss, and vermiculite is 2:1:1. The present invention preferably adds 1 / 2 MS nutrient solution to the seedling substrate in the early stage of transplanting to maintain appropriate humidity. In the present invention, the irrigation time with 1 / 2 MS nutrient solution can be 15 days. After irrigating with 1 / 2 MS nutrient solution for 15 days, the transplanted seedlings can be irrigated with water instead. As an optional embodiment of the present invention, during the seedling hardening and transplanting process, the cultivation temperature can be 24-26℃ or 25℃; the cultivation light intensity can be 1000-1500 lx, or 1000, 1100, 1200, 1300, 1400 or 1500 lx.

[0061] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0062] In the following technical solutions, BAP stands for 6-benzylaminopurine; 6-BA stands for 6-benzyladenine.

[0063] Example 1

[0064] 1. Screening of the most suitable disinfection method

[0065] Cut off stem segments with axillary buds from *Sinomenium acutum*, quickly rinse them with tap water, place them on a clean bench, rinse once with sterile water, soak in 75% ethanol solution for 30 seconds, rinse 3-4 times with sterile water, and disinfect with 2% NaClO solution for 3, 5, 7, and 10 minutes respectively (shaking continuously during this time). Label the four treatment numbers for different NaClO solution treatment times as A1-A4. After the NaClO solution treatment, repeat the washing with sterile water 3-4 times. Inoculate them into MS medium without exogenous hormones. The MS medium composition is: 4.74 g / L MS, 6.5 g / L agar, and 30 g / L sucrose. The pH of the MS medium is 5.8-6.0. Ten stem segments with axillary buds from *Sinomenium acutum* were inoculated for each treatment. Three parallel experiments were conducted for each treatment. The explants were placed in a culture room and cultured for 7 days at a temperature of 25℃, a light intensity of 1400 lx, and a light / dark cycle of 16 / 8 h. The number of surviving explants and the amount of contamination were recorded. The contamination rate and mortality rate of *Sinomenium acutum* explants were calculated. The results are shown in Table 1.

[0066] Table 1. Effects of NaClO disinfection time on contamination rate and mortality of explants.

[0067] A1 3 30 68.97 3.3 A2 5 30 7.4 10 A3 7 30 20.8 23.3 A4 10 30 0 93.3

[0068] Table 1 shows that the contamination rate of explants decreased with the extension of NaClO treatment time; at the same time, the explants turned black and the mortality rate increased significantly. When the NaClO treatment time was 5 min (A2 treatment), the contamination rate and mortality rate of explants were both low. Therefore, the disinfection method for using stem segments with axillary buds of *Sinomenium acutum* for tissue culture explants is as follows: first, soak in 75% ethanol solution for 30 s, wash 3-4 times with sterile water, disinfect with 2% NaClO solution for 5 min (with continuous shaking during the process), and finally rinse 3 times with sterile water.

[0069] Example 2

[0070] Screening of the optimal culture medium for inducing axillary bud differentiation into seedlings, i.e., screening of induction culture media.

[0071] See details for healthy stem segments of *Ipomoea purpurea* with axillary buds. Figure 1 Stem segments of *Sinomenium acutum* with axillary buds, 0.9–1.2 cm in length, were selected. The explants were sterilized using the optimal sterilization method selected in Example 1. They were then inoculated into induction media containing different ratios of 6-BA and NAA, and BAP and IAA. The basic composition of the induction media was 4.74 g / L MS, 6.5 g / L agar, and 30 g / L sucrose, with a pH of 5.8–6.0. ​​See the attached image for details of the sterilized *Sinomenium acutum* stem segments inoculated into the induction media. Figure 2The culture media with different ratios of 6-BA and NAA, and BAP and IAA, were labeled with six treatment numbers from B1 to B6, as shown in Table 2. Ten stem segments with axillary buds were inoculated into each culture medium, and three parallel experiments were conducted for each culture medium. The segments were placed in a culture room and induced to grow for 30 days under the conditions of 25℃, 1400 lx light intensity, and 16 / 8h (light / dark). The germination rate of the stem segments with axillary buds was counted, and the results are shown in Table 2.

[0072] Table 2 Effects of hormone concentration on axillary bud differentiation into seedlings

[0073]

[0074] Table 2 shows that four different concentrations of 6-BA and NAA combinations and two different concentrations of BAP and IAA combinations can all induce the differentiation of *Sinomenium acutum* stem segments with axillary buds. The emergence rate on the 2.0 mg / L 6-BA + 0.2 mg / L NAA medium reached 93.3%, significantly higher than other media, and is the optimal hormone ratio for inducing *Sinomenium acutum* stem segment differentiation with axillary buds. During the above experiments inducing axillary bud differentiation into seedlings, it was found that *Sinomenium acutum* is prone to browning. The emergence rate statistics in Table 2 are a statistical analysis of the emergence results of all treatment groups. Some experimental groups had poor growth after emergence, or even died during culture; these were also included in the emergence statistics. The emergence rate was calculated accordingly.

[0075] Example 3

[0076] Screening of anti-browning agents in induction culture medium

[0077] To alleviate browning of *Sinomenium acutum*, based on the optimal induction medium obtained in Example 2 (4.74 g / L MS + 2.0 mg / L 6-BA + 0.2 mg / L NAA + 6.5 g / L agar + 30 g / L sucrose, pH 5.8–6.0), different concentrations of activated carbon (Ac) and polyvinylpyrrolidone (PVP) were added to 1 L of induction medium, labeled C1–C5 as five treatment numbers (see Table 3 for details). Ten stem segments with axillary buds were inoculated into each medium, and three parallel experiments were conducted for each medium. The segments were placed in a culture room at 25°C, 1400 lx light intensity, and 16 / 8 h (light / dark) conditions for induction culture. The browning rate was calculated after 7 days of culture, and the mortality rate was calculated after 30 days. The results are shown in Table 3. After 30 days of culture, *Sinomenium acutum* tissue culture seedlings were obtained. Figure 3 As shown.

[0078] Table 3 Effects of different anti-browning agents on the induction of axillary bud seedling formation

[0079]

[0080] Table 3 shows that the addition of both Ac and PVP to the culture medium can inhibit explant browning. When the amount of Ac added reaches 1 g / L, it can effectively inhibit explant browning and better induce bud differentiation. The growth status of explants in the culture medium without Ac is shown in Table 3. Figure 4 As shown; the explant growth status when 0.1 g / L Ac is added to the culture medium is as follows. Figure 5 As shown; the explant growth status when 1 g / L Ac is added to the culture medium is as follows. Figure 6 As shown.

[0081] Example 4

[0082] Screening of the most suitable rooting culture medium

[0083] Tissue culture seedlings with good growth after 30 days of induction culture were selected from the induction media of experimental groups B1-B4 in Example 2 and experimental groups C1-C3 in Example 3. These seedlings were then transferred to rooting media composed of 1 / 2 MS, 6.5 g / L agar, 30 g / L sucrose, Ac, and different concentrations of NAA and IBA. The addition of Ac, NAA, and IBA to the rooting media is detailed in Table 4. The pH of the rooting media ranged from 5.8 to 6.0. Seven treatment numbers for different concentrations of NAA and IBA rooting media were labeled D1-D7. Ten healthy tissue culture seedlings were inoculated into each medium, and each medium was used in triplicate. The seedlings were placed in a culture room at 25°C, 1400 lx light, and a 16h / 8h day / night cycle for rooting culture. The rooting rate of the tissue culture seedlings was calculated after 30 days of culture. The results are shown in Table 4.

[0084] Table 4. Effects of hormone concentration on rooting of tissue culture seedlings

[0085]

[0086]

[0087] Table 4 shows that different concentrations of NAA and IBA in the rooting medium can induce rooting of tissue culture seedlings. The highest rooting rate was observed when the NAA concentration was 0.2 mg / L. Therefore, a medium consisting of 1 / 2 Ms + 1 g / L Ac + 6.5 g / L agar + 30 g / L sucrose with 0.2 mg / L NAA is the most suitable rooting medium for *Sinomenium acutum* tissue culture seedlings. After 30 days of cultivation in the above-mentioned optimal rooting medium, the rooted seedlings obtained are as follows: Figure 7 As shown.

[0088] Example 5

[0089] Hardening off and transplanting of rooted seedlings

[0090] Rooted seedlings from the D3 experimental group in Example 4 were cultured in the rooting medium for 30 days. After 5 days of semi-open culture, the seedlings were removed and transplanted into sterilized nutrient pots containing a mixture of potting soil, peat moss, and vermiculite in a volume ratio of 2:1:1. After transplanting, the seedlings were cultured with 1 / 2 MS nutrient solution to maintain appropriate humidity for 15 days, followed by watering. The culture temperature was 25℃, and the light intensity was 1400 lx. After 30 days of culture, the survival rate of the transplanted seedlings reached 99%.

[0091] Example 6

[0092] A tissue culture medium for the rapid propagation of *Sinomenium acutum*, consisting of an induction medium and a rooting medium.

[0093] The induction medium was based on MS medium, with the following added: 2 mg / L 6-BA, 0.2 mg / L NAA, 6.5 g / L agar and 30.0 g / L sucrose;

[0094] The rooting medium was based on 1 / 2 MS medium, with the following added ingredients: 1 g / L activated carbon, 0.2 mg / L NAA, 6.5 g / L agar, and 30.0 g / L sucrose.

[0095] Example 7

[0096] A tissue culture medium for the rapid propagation of *Sinomenium acutum*, consisting of an induction medium and a rooting medium.

[0097] The induction medium was based on MS medium, with the following added: 2 mg / L 6-BA, 0.1 mg / L NAA, 6.5 g / L agar and 30.0 g / L sucrose;

[0098] The rooting medium is based on 1 / 2 MS medium, and also contains: 1 g / L activated carbon, 0.2 mg / L NAA, 6.5 g / L agar and 30.0 g / L sucrose.

[0099] Example 8

[0100] A tissue culture medium for the rapid propagation of *Sinomenium acutum*, consisting of an induction medium and a rooting medium.

[0101] The induction medium was based on MS medium, with the following added: 1 mg / L 6-BA, 0.2 mg / L NAA, 6.5 g / L agar and 30.0 g / L sucrose;

[0102] The rooting medium is based on 1 / 2 MS medium, and also contains: 1 g / L activated carbon, 0.2 mg / L NAA, 6.5 g / L agar and 30.0 g / L sucrose.

[0103] Example 9

[0104] A tissue culture medium for the rapid propagation of *Sinomenium acutum*, consisting of an induction medium and a rooting medium.

[0105] The induction medium was based on MS medium, with the following added: 1 mg / L 6-BA, 0.1 mg / L NAA, 6.5 g / L agar and 30.0 g / L sucrose;

[0106] The rooting medium is based on 1 / 2 MS medium, and also contains: 1 g / L activated carbon, 0.2 mg / L NAA, 6.5 g / L agar and 30.0 g / L sucrose.

[0107] Example 10

[0108] A tissue culture medium for the rapid propagation of *Sinomenium acutum*, consisting of an induction medium and a rooting medium.

[0109] The induction medium was based on MS medium, supplemented with: 2 mg / L 6-BA, 0.2 mg / L NAA, 1 g / L LAc, 6.5 g / L agar and 30.0 g / L sucrose;

[0110] The rooting medium was based on 1 / 2 MS medium, with the following added ingredients: 1 g / L activated carbon, 0.2 mg / L NAA, 6.5 g / L agar, and 30.0 g / L sucrose.

[0111] Example 11

[0112] A tissue culture medium for the rapid propagation of *Sinomenium acutum*, consisting of an induction medium and a rooting medium.

[0113] The induction medium was based on MS medium, with the following added: 2 mg / L 6-BA, 0.2 mg / L NAA, 0.1 g / L activated charcoal, 6.5 g / L agar and 30.0 g / L sucrose;

[0114] The rooting medium was based on 1 / 2 MS medium, with the following added ingredients: 1 g / L activated carbon, 0.2 mg / L NAA, 6.5 g / L agar, and 30.0 g / L sucrose.

[0115] Example 12

[0116] A tissue culture medium for the rapid propagation of *Sinomenium acutum*, consisting of an induction medium and a rooting medium.

[0117] The induction medium was based on MS medium, with the following added: 2 mg / L 6-BA, 0.2 mg / L NAA, 1 g / L activated carbon, 6.5 g / L agar and 30.0 g / L sucrose;

[0118] The rooting medium was based on 1 / 2 MS medium, with the following added ingredients: 1 g / L activated carbon, 0.1 mg / L NAA, 6.5 g / L agar, and 30.0 g / L sucrose.

[0119] Example 13

[0120] A tissue culture medium for the rapid propagation of *Sinomenium acutum*, consisting of an induction medium and a rooting medium.

[0121] The induction medium was based on MS medium, with the following added: 2 mg / L 6-BA, 0.2 mg / L NAA, 1 g / L activated carbon, 6.5 g / L agar and 30.0 g / L sucrose;

[0122] The rooting medium was based on 1 / 2 MS medium, with the following added ingredients: 1 g / L activated carbon, 0.5 mg / L NAA, 6.5 g / L agar, and 30.0 g / L sucrose.

[0123] Example 14

[0124] A method for rapid propagation of *Ivy* (also known as Chinese artichoke) is as follows:

[0125] 1. Cut off stem segments with axillary buds from *Sinomenium acutum*, ensuring the length of each segment is 0.9–1.2 cm. Quickly rinse with tap water, place on a clean bench, rinse once with sterile water, soak in 75% ethanol solution for 30 seconds, rinse 3–4 times with sterile water, and disinfect with 2% NaClO solution for 5 minutes (shaking continuously during this time). After the NaClO solution treatment, repeat the washing process with sterile water 3–4 times to obtain the explants for inoculation, namely, *Sinomenium acutum* stem segments with axillary buds.

[0126] 2. Stem segments with axillary buds of *Sinomenium acutum* were inoculated into the induction medium of Example 6 for induction culture. Induction culture was conducted at 25°C, under 1400 lx light, and with a 16h / 8h day / night cycle for 30 days. After induction culture, induced seedlings were obtained.

[0127] 3. Select healthy seedlings and inoculate them into the rooting medium of Example 6 for rooting culture. Rooting culture is conducted at 25℃, under 1400 lx light, and with a day / night cycle of 16h / 8h for 30 days. After rooting culture is complete, rooted seedlings are obtained.

[0128] Example 15

[0129] The rooted seedlings obtained in Example 14 were selected for hardening-off and transplanting. The method is detailed in Example 5.

[0130] Example 16

[0131] A method for rapid propagation of *Sinomenium acutum* follows the same steps as in Example 14, except that the tissue culture medium used in Example 7 is employed for cultivation.

[0132] Example 17

[0133] A method for rapid propagation of *Sinomenium acutum* follows the same steps as in Example 14, except that the tissue culture medium used in Example 8 is employed for cultivation.

[0134] Example 18

[0135] A method for rapid propagation of *Sinomenium acutum* follows the same steps as in Example 14, except that the tissue culture medium used in Example 9 is employed for cultivation.

[0136] Example 19

[0137] A method for rapid propagation of *Sinomenium acutum* follows the same steps as in Example 14, except that the tissue culture medium used in Example 10 is employed for cultivation.

[0138] Example 20

[0139] A method for rapid propagation of *Sinomenium acutum* follows the same steps as in Example 14, except that the tissue culture medium used in Example 11 is employed for cultivation.

[0140] Example 21

[0141] A method for rapid propagation of *Sinomenium acutum* follows the same steps as in Example 14, except that the tissue culture medium used in Example 12 is employed for cultivation.

[0142] Example 22

[0143] A method for rapid propagation of *Sinomenium acutum* follows the same steps as in Example 14, except that the tissue culture medium used in Example 13 is employed for cultivation.

[0144] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for rapid propagation of *Ivy truncata*, characterized in that, include: Stem segments with axillary buds of *Sinomenium acutum* were induced and cultured in an induction medium to obtain induced seedlings. The induced seedlings were cultured in a rooting medium to obtain rooted seedlings; The induction medium is based on MS medium, and also contains: 2 mg / L 6-BA, 0.2 mg / L NAA, 1 g / L activated carbon, 6.5 g / L agar and 30.0 g / L sucrose; The rooting medium is based on 1 / 2 MS medium, and also contains: 1 g / L activated carbon, 0.2 mg / L NAA, 6.5 g / L agar and 30.0 g / L sucrose; The induction culture temperature is 24–26℃; the induction culture time is 28–32 days; and the light intensity during the induction culture is 1000–1500 lx. Before inoculating the stem segments with axillary buds of *Sinomenium acutum* into the induction medium, the process also includes sterilization of the stem segments with axillary buds. The disinfection method includes: After rinsing the stem segments of *Sinomenium acutum* with axillary buds, soak them in a 75% ethanol aqueous solution for 30 seconds, wash them 3-4 times with sterile water, disinfect them with a 2% sodium hypochlorite solution for 5 minutes, and finally rinse them 3 times with sterile water.

2. The method according to claim 1, characterized in that, The rooting culture temperature is 24–26℃; the rooting culture time is 12–30 days; and the light intensity during the rooting culture is 1000–1500 lx.

3. The method according to claim 1, characterized in that, The stem segment with axillary buds has a length of 0.9–1.2 cm.

4. The method according to claim 1, characterized in that, The method further includes: hardening off the rooted seedlings and transplanting them.

5. The application of the method according to any one of claims 1 to 4 in the rapid propagation of *Sinomenium acutum*.

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