Tissue culture medium for rapid propagation of caulis sinomenii and method for rapid propagation of caulis sinomenii

Abstract

The invention 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 invention provides a tissue culture medium for rapid propagation of caulis sinomenii. The tissue culture medium comprises an induction medium and a rooting medium, the induction culture medium takes an MS culture medium as a basic culture medium, and further comprises 1-2mg / L of 6-BA and 0.1-0.2 mg / L of NAA; the rooting culture medium takes a 1 / 2 MS culture medium as a basic culture medium, and further comprises 0.1-1g / L of LAc and 0.1-0.2 mg / L of NAA. According to the embodiment of the invention, stem segments with buds of caulis sinomenii are taken as explants, axillary buds are directly induced to differentiate by utilizing the tissue culture medium, a large number of seedlings can be obtained, mass propagation can be realized within a short time, and the propagation efficiency of caulis sinomenii is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and particularly relates to a tissue culture medium for rapid propagation of Caulis Sinomenii and a method for rapid propagation of Caulis Sinomenii. Background Art

[0002] Sinomenium sinomenium is a plant of the Menispermaceae family. It has the effects of dispelling rheumatism, unblocking meridians and promoting 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 more and more attention from domestic and foreign scholars and medical commercial development and application, and the demand is constantly increasing. However, Sinomenium sinomenium is usually harvested with the roots, and the wild resource population of Sinomenium sinomenium is in decline. In addition, Sinomenium sinomenium is a perennial woody vine with a long growth period. When cultivated in the field, it is easily affected by many adverse factors such as differences in production areas, climate change, pest and disease invasion, heavy metal and pesticide residues in the soil, etc., resulting in the uneven quality of Sinomenium sinomenium medicinal materials sold on the market, and the consequences such as poor clinical treatment effects of related pharmaceutical preparations have made it difficult to meet the increasingly stringent quality requirements of traditional Chinese medicines in the domestic and foreign markets.

[0003] Plant tissue culture can produce high-quality seedlings that are genetically consistent, pest-free, and have strong growth potential in a short period of time, ensuring the stability of excellent traits and achieving large-scale, year-round production of high-quality plants. Therefore, for Caulis Sinomenii, using plant tissue culture methods to expand the reproduction of Caulis Sinomenii and cultivate strong seedlings for Caulis Sinomenii is of great significance for improving its medicinal quality. However, to date, there are no reports in the prior art on biotechnology related to rapid propagation of Caulis Sinomenii by tissue culture. Summary of the Invention

[0004] In view of the defects in the prior art, the object of the present invention is to provide a tissue culture medium for the rapid propagation of Caulis Sinomenii, which can realize the rapid propagation of Caulis Sinomenii using the stem segments of Caulis Sinomenii with axillary buds.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] The present invention provides a tissue culture medium for rapid propagation of Caulis Sinomenii, comprising an induction medium and a rooting medium;

[0007] The induction culture medium is based on MS culture 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;

[0008] The rooting culture medium uses 1 / 2 MS culture medium as the basic culture 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.

[0009] Preferably, the induction medium further comprises 0.1-1 g / L activated carbon.

[0010] Preferably, the induction medium is based on MS medium and further comprises: 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 culture medium uses 1 / 2 MS culture medium as the basic culture medium and further comprises: 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] The present invention provides application of the tissue culture medium described in the above technical solution in the rapid propagation of Sinomenium sinomenium.

[0013] The present invention provides a method for rapid propagation of Sinomenium sinomenium, based on the tissue culture medium described in the above technical solution, comprising:

[0014] Inducing and culturing the stem segments of Caulis Sinensis with axillary buds in the induction medium to obtain induced seedlings;

[0015] The induced seedlings are subjected to rooting culture in the rooting medium to obtain rooted seedlings.

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

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

[0018] Preferably, the length of the stem segment with axillary buds is 0.9 to 1.2 cm.

[0019] Preferably, before inoculating the stem segments of Caulis Achyranthis Bidens with axillary buds into the induction culture medium, the method further comprises disinfecting the stem segments of Caulis Achyranthis Bidens with axillary buds;

[0020] The method of disinfection comprises:

[0021] The stem segments of Caulis Sinomenii with axillary buds are rinsed and then soaked in an ethanol aqueous solution and a sodium hypochlorite aqueous solution in sequence; the soaking time in the sodium hypochlorite aqueous solution is 4 to 7 minutes.

[0022] Preferably, the method further comprises: hardening and transplanting the rooted seedlings.

[0023] Beneficial effects of the present invention:

[0024] The invention provides a tissue culture medium for rapid propagation of Caulis Sinomenii, comprising an induction medium and a rooting medium; the induction medium uses an MS medium as a base 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 uses a 1 / 2 MS medium as a base 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. In the tissue culture medium provided by the present invention, 6-BA (6-benzylaminopurine) in the induction medium is a cytokinin that can promote cell division and expansion and induce bud formation; NAA (naphthaleneacetic acid) is an auxin that can promote the growth and differentiation of axillary buds of Caulis et Rhizoma Coptidis; the combined action of 6-BA and NAA is conducive to inducing the germination of primary clustered buds from the stem segments with axillary buds of Caulis et Rhizoma Coptidis, thereby increasing the proliferation coefficient of clustered buds; Ac (activated carbon) has good adsorption properties and can adsorb harmful substances in the culture medium, including impurities contained in agar, phenols, quinones, and other substances that are harmful to the explants themselves, such as phenols and quinones secreted by the explants during the culture process. 5-Hydroxymethylfurfural produced by sucrose during high-pressure sterilization is beneficial to the differentiation and proliferation of buds. Adding NAA and Ac to the rooting medium can induce root formation and promote the growth of the root system of tissue culture seedlings. The combined action of NAA and Ac can increase the rooting rate. The results of the embodiment show that using the stem segments with buds of Caulis et Rhizoma Coptidis as explants, axillary bud differentiation is directly induced, a large number of subculture seedlings are obtained, and a large number of seedlings can be propagated in a relatively short period of time. Compared with other tissue culture methods, callus culture is not required, the steps are reduced, and the propagation efficiency of Caulis Sinomenii is improved. The tissue culture medium provided by the present invention is used for Caulis Sinomenii tissue culture, and has the technical characteristics of high seedling rate, simple operation, short growth cycle and high technical practicality. In summary, the tissue culture medium provided by the present invention can significantly improve the survival rate of tissue culture seedlings through the combination of various components in the induction medium and the rooting medium, thereby achieving the purpose of rapid propagation of Caulis Sinomenii. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 This is a photo of a healthy stem segment of Caulis Sinensis with axillary buds;

[0027] Figure 2 The figure shows the stem segment with axillary buds of Caulis Cibotii being sterilized and inoculated into the induction medium for cultivation;

[0028] Figure 3 This is a photo of the tissue culture seedlings (induced seedlings) of Caulis Sinomenii that were generated through induced culture;

[0029] Figure 4 This is a graph showing the growth status of explants when Ac was not added to the induction medium;

[0030] Figure 5 This is a graph showing the growth status of explants when 0.1 g / LAc was added to the induction medium;

[0031] Figure 6 This is a graph showing the growth status of explants when 1 g / LAc was added to the induction medium;

[0032] Figure 7 This is a photo of the rooted seedlings that have undergone rooting culture;

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

[0034] The present invention provides a tissue culture medium for rapid propagation of Caulis Sinomenii, comprising an induction medium and a rooting medium;

[0035] The induction culture medium is based on MS culture 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;

[0036] The rooting culture medium uses 1 / 2 MS culture medium as the basic culture 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.

[0037] In the following technical solutions, the present invention has no special restrictions on the sources of the raw materials, and any conventional commercially available products in the field can be used.

[0038] In the present invention, the tissue culture medium includes an induction medium. In the present invention, the induction medium uses MS medium as a basal medium. In the present invention, the MS medium can be added in an amount of 4.74 g / L. In the present invention, the MS medium can meet the nutritional requirements of tissue culture seedlings germinated from axillary buds of Caulis Cibotii and promote the growth of induced seedlings.

[0039] In the present invention, the induction medium includes 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 the present invention, the 6-BA in the induction medium can promote cell division and expansion, and induce bud formation.

[0040] In the present invention, the induction 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 the present invention, the NAA in the induction medium can promote the growth and differentiation of the stem segments with axillary buds of Caulis Sinensis.

[0041] In the present invention, the induction culture medium includes 6.0-7.0 g / L agar, and may also be 6.5 g / L.

[0042] In the present invention, the induction medium comprises 30.0-30.5 g / L sucrose, and may also be 30.0 g / L. In the present invention, 5-hydroxymethylfurfural produced by the sucrose in the induction medium during autoclaving is beneficial to the differentiation and proliferation of buds.

[0043] In the present invention, the induction medium preferably further comprises 0.1 to 1 g / L of activated carbon, which may be 0.5 to 1 g / L, or may be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 g / L. In the present invention, the activated carbon in the induction medium has good adsorption properties and can adsorb harmful substances in the culture medium, including impurities contained in agar, phenols and quinones secreted by the explants during the culture process, and other substances that are harmful to the explants themselves, thereby helping to reduce the occurrence of browning problems in the explants and further improve the emergence rate.

[0044] In the present invention, the pH value of the induction medium may be 5.8-6.0, or may 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 Caulis Sinensis, improve the proliferation coefficient of clustered buds, inhibit the browning of explants, and thus is conducive to improving the seedling emergence rate of the stem segments with axillary buds of Caulis Sinensis.

[0046] In the present invention, the tissue culture medium includes a rooting medium. In the present invention, the rooting medium is based on 1 / 2 MS medium. In the present invention, the 1 / 2 MS medium is specifically a commercially available MS medium with an addition amount of 2.37 g / L. In the present invention, the 1 / 2 MS medium can meet the nutritional requirements of the rooting of the tissue culture seedlings of Qingfengteng, promoting the growth of the tissue culture seedlings while promoting the elongation of the roots of the tissue culture seedlings.

[0047] In the present invention, the rooting medium includes 0.1-1 g / L activated carbon, which may 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 the present invention, the activated carbon in the rooting medium can absorb harmful substances, reduce browning, and promote root development and increase the rooting rate.

[0048] In the present invention, the rooting medium includes 0.1-0.2 mg / L NAA, which may 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 the present invention, the addition of NAA to the rooting medium can induce root formation in tissue culture seedlings, promote root growth, and help improve the rooting rate.

[0049] In the present invention, the rooting medium includes 6.0-7.0 g / L agar, and may also be 6.5 g / L.

[0050] In the present invention, the rooting medium includes 30.0-30.5 g / L sucrose, and may also be 30 g / L.

[0051] In the present invention, the pH value of the rooting medium may be 5.8-6.0, or may be 5.8, 5.9 or 6.0.

[0052] Adding NAA and activated carbon to the rooting medium described in the above technical solution of the present invention can promote the growth of the root system of tissue culture seedlings, and the combined action of NAA and activated carbon can increase the rooting rate.

[0053] The present invention provides the use of the tissue culture medium described in the above technical solution for the rapid propagation of Caulis Sinomenii. The tissue culture medium provided by the present invention can induce the differentiation of Caulis Sinomenii axillary buds into complete plants, improve the emergence rate and rooting rate of tissue culture seedlings, and thus achieve rapid propagation of Caulis Sinomenii. The present invention uses bud-bearing stem segments of Caulis Sinomenii as explants and adopts the tissue culture medium to directly induce axillary bud differentiation and rooting, thereby obtaining a large number of rooted seedlings, and can achieve large-scale seedling propagation in a relatively short period of time.

[0054] The present invention provides a method for rapid propagation of Sinomenium sinomenium, based on the tissue culture medium described in the above technical solution, comprising:

[0055] Inducing and culturing the stem segments of Caulis Sinensis with axillary buds in the induction medium to obtain induced seedlings;

[0056] The induced seedlings are subjected to rooting culture in the rooting medium to obtain rooted seedlings.

[0057] The present invention induces and cultures the stem segments of Caulis dasyphylla with axillary buds in the induction culture medium to obtain induced seedlings. As an optional embodiment of the present invention, the stem segments with axillary buds can be 0.9 to 1.2 cm in length, or can be 0.9, 1.0, 1.1, or 1.2 cm. In the present invention, the stem segments of Caulis dasyphylla with axillary buds preferably include healthy stem segments of Caulis dasyphylla with axillary buds; preferably, each of the stem segments of Caulis dasyphylla with axillary buds has one axillary bud. After obtaining the stem segments of Caulis dasyphylla with axillary buds, the present invention preferably disinfects the stem segments of Caulis dasyphylla with axillary buds. As an optional embodiment of the present invention, the disinfection method comprises: rinsing the stem segments of Caulis dasyphylla with axillary buds and then soaking them in an ethanol aqueous solution and a sodium hypochlorite aqueous solution, sequentially; the soaking time in the sodium hypochlorite aqueous solution is 4 to 7 minutes. The present invention does not specifically limit the rinsing method; any conventional rinsing method in the art can be used. As an optional embodiment of the present invention, the rinsing method can be rinsing with tap water followed by another rinse with sterile water. After rinsing, the present invention preferably soaks the Caulis Australis stem segments with axillary buds in an ethanol-water solution; the volume fraction of ethanol in the ethanol-water solution can be 75% and the soaking time can be 30 seconds. After soaking in the ethanol-water solution, the present invention preferably rinses the Caulis Australis stem segments with axillary buds 3-4 times with sterile water and then soaks them in an aqueous sodium hypochlorite solution; the mass fraction of sodium hypochlorite in the aqueous sodium hypochlorite solution is 2%; the soaking time can be 4-7 minutes, or 4, 5, 6, or 7 minutes. The present invention preferably shakes the Caulis Australis stem segments continuously during the sodium hypochlorite soaking process. After the sodium hypochlorite soaking, the present invention preferably rinses the Caulis Australis stem segments with axillary buds 3-4 times with sterile water. After the sterile water rinse, the present invention inoculates the resulting Caulis Australis stem segments with axillary buds into an induction culture medium for induction culture.

[0058] As an optional embodiment of the present invention, the temperature of the induction culture can be 24-26°C, or 25°C; the light intensity of 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 obtains induced seedlings.

[0059] After obtaining the induced seedlings, the present invention performs rooting culture on the induced seedlings in the rooting medium to obtain rooted seedlings. As an optional embodiment of the present invention, the temperature of the rooting culture can be 24-26°C, or 25°C; the light intensity of the rooting culture can be 1000-1500 lx, or 1000, 1100, 1200, 1300, 1400, or 1500 lx; ​​the time of the rooting culture 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 rooted seedlings, the present invention preferably further comprises hardening and transplanting the rooted seedlings. The present invention does not specifically limit the methods for hardening and transplanting; conventional hardening and transplanting methods in the art may be employed. As an optional embodiment of the present invention, the hardening method comprises: culturing the rooted seedlings in a rooting medium with a semi-open lid; the semi-open lid culturing period may be 5 days. After the semi-open lid culturing period is completed, the present invention preferably transplants the resulting tissue culture seedlings. The present invention preferably transplants the tissue culture seedlings in a seedling culture medium. As an optional embodiment of the present invention, the seedling culture medium comprises sterilized nutrient soil, peat, and vermiculite; the volume ratio of the nutrient soil, peat, and vermiculite is 2:1:1. The present invention preferably adds 1 / 2MS nutrient solution to the seedling culture medium before transplanting to maintain appropriate humidity. In the present invention, irrigation with the 1 / 2MS nutrient solution may be performed for 15 days. After 15 days of irrigation with the 1 / 2MS nutrient solution, the transplanted seedlings may be watered with water. As an optional embodiment of the present invention, during the process of hardening and transplanting, the culture temperature can be 24-26°C, or 25°C; the culture light intensity can be 1000-1500lx, or 1000, 1100, 1200, 1300, 1400 or 1500lx.

[0061] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

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

[0063] Example 1

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

[0065] The stem segments with axillary buds of Caulis Sinomenii were cut off, quickly rinsed with tap water, placed on a clean bench, rinsed once with sterile water, soaked in 75% ethanol solution for 30 seconds, washed with sterile water 3 to 4 times, and disinfected with 2% NaClO solution for 3, 5, 7, and 10 minutes (shaken continuously during the period). The four treatment numbers of different NaClO solution treatment times were marked as A1 to A4. After the NaClO solution treatment, the segments were washed repeatedly with sterile water 3 to 4 times and inoculated into MS medium without exogenous hormones. The MS medium composition was: 4.74 g / L MS, 6.5 g / L agar, and 30 g / L sucrose. The pH value of the MS medium was 5.8-6.0. Ten stem segments with axillary buds of Caulis Sinomenii were inoculated in each treatment, and three parallel experiments were conducted for each treatment. The stem segments were placed in a culture room and cultured for 7 days at a temperature of 25°C, a light intensity of 1400 lx, and a 16 / 8 h (light / dark) cycle. The number of surviving explants and the number of contaminated explants were recorded, and the contamination rate and mortality rate of Caulis Sinomenii explants were calculated. The results are shown in Table 1.

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

[0067] Test number Disinfection time, min Number of explants Contamination rate, % mortality rate,% 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] As shown in Table 1, the contamination rate of explants decreased with increasing NaClO treatment time; at the same time, the explants turned black and their mortality rate increased significantly. When the NaClO treatment time was 5 minutes (treatment A2), both the contamination rate and mortality rate of the explants were low. Therefore, the disinfection method for tissue culture explants using stem segments of Caulis et Rhizoma with axillary buds was as follows: first, soak in 75% ethanol solution for 30 seconds, rinse with sterile water three to four times, disinfect in 2% NaClO solution for 5 minutes (shaking continuously), and finally rinse three 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 medium

[0071] See the healthy stem segments of Qingfengteng with axillary buds for details. Figure 1 , select the stem segments of Caulis Australis with axillary buds, the length of the stem segments is 0.9 to 1.2 cm. Use the optimal disinfection method selected in Example 1 for disinfection, and then inoculate the explants in an induction medium containing different ratios of 6-BA and NAA, BAP and IAA. The basic composition of the induction medium is 4.74g / LMS, 6.5g / L agar and 30g / L sucrose, and the pH value of the induction medium is 5.8 to 6.0. After disinfection, the stem segments of Caulis Australis with axillary buds are inoculated in the induction medium for culture. See the figure for details. Figure 2, 6-BA and NAA, and BAP and IAA in different ratios were used as culture media, designated B1 to B6, for treatments (see Table 2). Ten stem segments with axillary buds from C. chinensis were inoculated on each culture medium, with three replicates conducted on each medium. These were placed in a culture room and induced for 30 days at 25°C, 1400 lx light intensity, and a 16 / 8 h light / dark cycle. The emergence rate of the stem segments with axillary buds was calculated, as shown in Table 2.

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

[0073]

[0074] As shown in Table 2, four combinations of 6-BA and NAA at different concentrations and two combinations of BAP and IAA at different concentrations can induce the differentiation of stem segments with axillary buds of Caulis Achyranthis Bidentatae. Among them, the germination rate on the 2.0 mg / L6-BA+0.2 mg / LNAA culture medium reached 93.3%, which was significantly higher than other culture media. It is the best hormone ratio for inducing the differentiation of stem segments with axillary buds of Caulis Achyranthis Bidentatae. In the above-mentioned experiment of inducing axillary bud differentiation into seedlings, it was found that Caulis Achyranthis Bidentatae is prone to browning. The statistics of the germination rate in Table 2 are statistics of all the results of germination in each treatment group. Some experimental groups had poor growth after germination, and even died during the culture process, which were also included in the statistical results of germination. Calculation of germination rate was performed.

[0075] Example 3

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

[0077] In order to alleviate the browning of Caulis Adenophorae, based on the optimal induction medium screened in Example 2, i.e. 4.74g / LMS+2.0mg / L 6-BA+0.2mg / LNAA+6.5g / L agar+30g / L sucrose, pH=5.8~6.0, different concentrations of activated carbon (Ac) and polyvinyl pyrrolidone (PVP) were added to 1L induction medium, and 5 treatment numbers were marked with C1~C5 respectively. For details, see Table 3. Ten Caulis Adenophorae stem segments with axillary buds were inoculated into each culture medium, and 3 parallel experiments were conducted for each culture medium. They were placed in a culture room and induced at a temperature of 25°C, a light intensity of 1400lx, and 16 / 8h (light / dark). The browning rate was counted after 7 days of culture, and the mortality rate was counted after 30 days of culture. The results are shown in Table 3. After 30 days of culture, Caulis Adenophorae tissue culture seedlings were obtained, as shown in Table 3. Figure 3 shown.

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

[0079]

[0080] As shown in Table 3, the addition of Ac and PVP to the culture medium can inhibit the browning of the explants. When the amount of Ac added reaches 1 g / L, it can not only effectively inhibit the browning of the explants, but also better induce the differentiation of buds. Figure 4 As shown; when 0.1g / LAc was added to the culture medium, the growth of the explants was as follows Figure 5 When 1 g / L Ac was added to the culture medium, the growth of the explants was as shown in Figure 6 shown.

[0081] Example 4

[0082] Screening of the optimal rooting medium

[0083] Tissue culture seedlings that had grown well after 30 days of induction culture in the induction medium for experimental groups B1-B4 in Example 2 and C1-C3 in Example 3 were selected and transferred to a rooting medium composed of 1 / 2 MS, 6.5 g / L agar, 30 g / L sucrose, Ac, and varying concentrations of NAA and IBA. The addition of Ac, NAA, and IBA to the rooting medium is detailed in Table 4. The pH of the rooting medium was 5.8-6.0. Seven rooting medium treatments with varying concentrations of NAA and IBA were designated D1-D7. Ten well-grown tissue culture seedlings were inoculated into each medium. Three replicates were run for each medium. The seedlings were placed in a culture room and cultured for rooting under the following conditions: a temperature of 25°C, a light intensity of 1400 lx, and a 16 / 8 hour cycle. The rooting rate of the tissue culture seedlings was calculated after 30 days of incubation. The results are shown in Table 4.

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

[0085]

[0086]

[0087] As shown in Table 4, different concentrations of NAA and IBA in the rooting medium can induce rooting of tissue culture seedlings. When the NAA concentration is 0.2 mg / L, the rooting rate of tissue culture seedlings is the highest. Therefore, the medium composed of 1 / 2Ms+1g / LAc+6.5g / L agar+30g / L sucrose with 0.2 mg / L NAA is the most suitable rooting medium for tissue culture seedlings of Qingfengteng. After 30 days of cultivation in the above optimal rooting medium, the rooted seedlings obtained are as follows: Figure 7 shown.

[0088] Example 5

[0089] Hardening and transplanting of rooted seedlings

[0090] The D3 test group in the embodiment 4 was selected to cultivate 30d in the rooting medium. The rooted seedlings with consistent growth were taken out after half-opening the cover to cultivate for 5d and transplanted into a sterilized nutrient pot with a mixed matrix of nutrient soil, peat and vermiculite, wherein the volume ratio of nutrient soil, peat and vermiculite was 2:1:1. After transplanting, the seedlings were watered after maintaining appropriate humidity for 15d with 1 / 2MS nutrient solution. The cultivation temperature was 25°C and the illumination was 1400lx. After cultivating for 30 days, the survival rate of the transplanted seedlings could reach 99%.

[0091] Example 6

[0092] A tissue culture medium for rapid propagation of Caulis Sinomenii, comprising an induction medium and a rooting medium.

[0093] The induction medium was based on MS medium supplemented with: 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 is based on 1 / 2MS medium, and is supplemented with: 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 rapid propagation of Caulis Sinomenii, comprising an induction medium and a rooting medium.

[0097] The induction medium was based on MS medium supplemented with: 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 is further supplemented with: 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 rapid propagation of Caulis Sinomenii, comprising an induction medium and a rooting medium.

[0101] The induction medium was based on MS medium supplemented with: 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 is further supplemented with: 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 rapid propagation of Caulis Sinomenii, comprising an induction medium and a rooting medium.

[0105] The induction medium was based on MS medium supplemented with: 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 is further supplemented with: 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 rapid propagation of Caulis Sinomenii, comprising 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 Ac, 6.5 g / L agar, and 30.0 g / L sucrose;

[0110] The rooting medium is based on 1 / 2MS medium, and is supplemented with: 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 rapid propagation of Caulis Sinomenii, comprising an induction medium and a rooting medium.

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

[0114] The rooting medium is based on 1 / 2MS medium, and is supplemented with: 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 rapid propagation of Caulis Sinomenii, comprising an induction medium and a rooting medium.

[0117] The induction medium was based on MS medium supplemented with: 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 is based on 1 / 2 MS medium, and is supplemented with: 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 rapid propagation of Caulis Sinomenii, comprising an induction medium and a rooting medium.

[0121] The induction medium was based on MS medium supplemented with: 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 is based on 1 / 2 MS medium, and is supplemented with: 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 Caulis Sinomenii, comprising the following steps:

[0125] 1. Cut the stem segments with axillary buds of Caulis Sinomenii to a length of 0.9-1.2 cm, rinse quickly with tap water, place on a clean bench, rinse once with sterile water, soak in 75% ethanol solution for 30 seconds, wash 3-4 times with sterile water, and disinfect with 2% NaClO solution for 5 minutes (shaking continuously during the period). After the NaClO solution treatment is completed, wash with sterile water 3-4 times to obtain the explants for inoculation, i.e., the stem segments with axillary buds of Caulis Sinomenii.

[0126] 2. The stem segments of Caulis Sinomenii with axillary buds were inoculated into the induction medium of Example 6 for induction culture at 25°C, 1400 lx of light, and 16h / 8h of day and night. The induction culture lasted for 30 days. After the induction culture was completed, induced seedlings were obtained.

[0127] 3. Select the induced seedlings with good growth and inoculate them into the rooting medium of Example 6 for rooting culture. The rooting culture was carried out under the conditions of temperature 25°C, light intensity 1400 lx, and day and night 16h / 8h. The rooting culture time was 30 days. After the rooting culture was completed, rooted seedlings were obtained.

[0128] Example 15

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

[0130] Example 16

[0131] A method for rapid propagation of Sinomenium sinomenium, comprising the same steps as in Example 14, with the only difference being that the tissue culture medium of Example 7 is used for culturing.

[0132] Example 17

[0133] A method for rapid propagation of Sinomenium sinomenium, comprising the same steps as in Example 14, with the only difference being that the tissue culture medium of Example 8 is used for culturing.

[0134] Example 18

[0135] A method for rapid propagation of Sinomenium sinomenium, comprising the same steps as in Example 14, with the only difference being that the tissue culture medium of Example 9 is used for culturing.

[0136] Example 19

[0137] A method for rapid propagation of Sinomenium sinomenium, comprising the same steps as in Example 14, with the only difference being that the tissue culture medium of Example 10 is used for culturing.

[0138] Example 20

[0139] A method for rapid propagation of Sinomenium sinomenium, comprising the same steps as in Example 14, with the only difference being that the tissue culture medium of Example 11 is used for culturing.

[0140] Example 21

[0141] A method for rapid propagation of Sinomenium sinomenium, comprising the same steps as in Example 14, with the only difference being that the tissue culture medium of Example 12 is used for culturing.

[0142] Example 22

[0143] A method for rapid propagation of Sinomenium sinomenium, comprising the same steps as in Example 14, with the only difference being that the tissue culture medium of Example 13 is used for culturing.

[0144] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A tissue culture medium for rapid propagation of Caulis Sinomenii, characterized in that: Includes induction medium and rooting medium; The induction culture medium is based on MS culture 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 culture medium uses 1 / 2 MS culture medium as the basic culture 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.

2. The tissue culture medium according to claim 1, wherein The induction culture medium also includes 0.1-1 g / L activated carbon.

3. The tissue culture medium according to claim 1 or 2, characterized in that 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; The rooting culture medium uses 1 / 2 MS culture medium as the basic culture medium and further comprises: 0.5-1 g / L activated carbon, 0.15-0.2 mg / L NAA, 6.5 g / L agar and 30 g / L sucrose.

4. Use of the tissue culture medium according to any one of claims 1 to 3 in the rapid propagation of Caulis Sinomenii.

5. A method for rapid propagation of Caulis Sinomenii, characterized in that: The tissue culture medium according to any one of claims 1 to 3, comprising: Inducing and culturing the stem segments of Caulis Sinensis with axillary buds in the induction medium to obtain induced seedlings; The induced seedlings are subjected to rooting culture in the rooting medium to obtain rooted seedlings.

6. The method according to claim 5, characterized in that The induction culture temperature is 24-26° C.; the induction culture time is 28-32 days.

7. The method according to claim 5, characterized in that The temperature of the rooting culture is 24-26° C.; the time of the rooting culture is 12-30 days.

8. The method according to claim 5, characterized in that The length of the stem segment with axillary buds is 0.9 to 1.2 cm.

9. The method according to claim 5, characterized in that: Before inoculating the stem segment with axillary buds of Caulis Sinensis into the induction culture medium, the method further includes disinfecting the stem segment with axillary buds of Caulis Sinensis; The method of disinfection comprises: The stem segments of Caulis Sinomenii with axillary buds are rinsed and then soaked in an ethanol aqueous solution and a sodium hypochlorite aqueous solution in sequence; the soaking time in the sodium hypochlorite aqueous solution is 4 to 7 minutes.

10. The method according to claim 5, characterized in that: The method further comprises: hardening and transplanting the rooted seedlings.

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