Efficient in vitro rapid propagation method of dioscorea composita

Abstract

The present application belongs to the technical field of medicinal plant tissue culture. More specifically, the present application provides a method for in vitro rapid propagation of D. japonica, wherein a stem section with buds of D. japonica is inoculated into an absorption surface induction medium for absorption surface induction, and the absorption surface induction medium comprises MS medium and thidiazuron (TDZ). The present application uses the stem section with buds of D. japonica as explants, induces the generation of swollen absorption surface, and enhances the adaptability of the explants to the tissue culture environment. The present application overcomes the vitrification and browning phenomena of the tissue culture seedlings of D. japonica in the cultivation process, and does not require multiple subcultures to stabilize the growth of the tissue culture seedlings, thereby improving the proliferation and expansion speed of the tissue culture seedlings and shortening the culture time of the tissue culture seedlings. In addition, the tissue culture seedlings produced by the in vitro rapid propagation method of D. japonica have a unified genotype, a small variation probability, and are easy to standardize during the breeding process. The present application can be applied to the stable production of high-quality seedlings of D. japonica, and has good application prospects and value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicinal plant tissue culture. More particularly, it relates to a high-efficiency in-vitro rapid propagation method of Dioscorea composita. BACKGROUND

[0002] Dioscorea composita Hemsl., also known as Mexican yam, belongs to the Dioscoreaceae family and is a perennial climbing herb. It is mainly distributed in the tropical and subtropical regions of Asia and America. It has multiple economic values, such as extracting diosgenin and other dioscorea saponins. Diosgenin, also known as saponin, is a natural secondary metabolite with multiple pharmacological effects. It can be used for the prevention and treatment of cancer, asthma, diabetes, arthritis, cardiovascular disease, hypertension, coronary heart disease, and vascular sclerosis. It is an important raw material for the synthesis of more than 300 steroid hormone drugs such as adrenocortical hormones and sex hormones, and is known as "medicinal gold". Currently, most of the corticosteroids, sex hormones, and protein anabolic hormones produced worldwide are based on diosgenin as the basic raw material, which has extremely high economic and medicinal value. Therefore, the demand for diosgenin is very large. Therefore, how to efficiently and massively propagate Dioscorea composita to meet the future market and development needs of diosgenin is of great significance.

[0003] Currently, the conventional cultivation methods of Dioscorea composita mainly include seed propagation and rhizome propagation. Although seed propagation has the advantages of relatively simple operation and low cost, it is greatly affected by seasonal, climatic, and environmental factors, and the seed propagation process in China also has the phenomenon of difficulty in seed setting, which seriously limits the propagation rate and has certain limitations in large-scale production. Rhizome propagation is limited by the long growth cycle, low underground rhizome propagation coefficient, high cost, and large amount of seed used, making it difficult to achieve rapid popularization and planting. Therefore, the existing Dioscorea composita seedling propagation speed does not match the market demand, affecting the development of the Dioscorea composita industry.

[0004] Plant tissue culture and rapid propagation is an important technical means commonly used in seedling propagation process, and has been widely used in fruit and vegetable, landscape trees, forest trees, economic crops and medicinal plants production fields. The application of plant tissue culture and rapid propagation significantly improves the plant seedling production efficiency, expands the plant seedling yield, promotes the plant seedling factory production, and brings huge economic and social benefits. However, the current tissue culture and rapid propagation of D. japonica needs multiple subculture to stabilize the growth of tissue culture seedlings, and the tissue culture seedlings are prone to vitrification or browning in multiple subculture. That is, after the disinfection and primary culture of the explant of D. japonica, multiple subculture is needed to continue the next step of adventitious bud induction and elongation culture and rooting culture, resulting in low and unstable tissue culture and rapid propagation efficiency of D. japonica.

[0005] Therefore, it is of great significance to research and develop high-efficiency in vitro rapid propagation technology of D. japonica for promoting the factory production of D. japonica tissue culture and rapid propagation seedlings. SUMMARY

[0006] In order to solve the problems of vitrification or browning of D. japonica tissue culture seedlings and the need for multiple subculture to stabilize the growth of tissue culture seedlings, the application provides a method for in vitro rapid propagation of D. japonica.

[0007] The above-mentioned object of the application is achieved by the following technical scheme:

[0008] After a large amount of research and exploration, the application develops a culture medium capable of inducing the stem segment (explant) of D. japonica with buds to form an enlarged spherical structure (named absorption surface) at the axillary bud in contact with the culture medium, which is named absorption surface induction medium and absorption surface expansion medium. After disinfection and primary culture of the stem segment (explant) of D. japonica, subculture is no longer needed, and the absorption surface induction medium and absorption surface expansion medium are used for culture, and then the next step of adventitious bud induction and elongation culture and rooting culture is directly carried out. This technology not only omits the multiple subculture link, shortens the tissue culture time, but also overcomes the vitrification and browning of D. japonica tissue culture seedlings in the cultivation process.

[0009] Therefore, the application provides the application of MS medium containing thidiazuron and MS medium containing 6-benzylaminopurine in in vitro rapid propagation of D. japonica.

[0010] The MS medium containing thidiazuron, i.e. absorption surface induction medium, wherein the concentration of thidiazuron is 1.0-1.5 mg / L.

[0011] The MS medium containing 6-benzylaminopurine, i.e. absorption surface expansion medium, wherein the concentration of 6-benzylaminopurine is 3-7 mg / L.

[0012] As an alternative embodiment, the absorption surface induction medium further comprises sucrose and agar.

[0013] Optionally, the absorption surface induction medium comprises the following components: MS medium, 1.0-1.5 mg / L thidiazuron, 1-5% sucrose, and 0.1-1% agar.

[0014] Optionally, the absorption surface induction medium comprises the following components: MS medium, 1.0-1.5 mg / L thidiazuron, 3% sucrose, and 0.7% agar.

[0015] Optionally, the absorption surface induction medium comprises the following components: MS medium, 1.0-1.5 mg / L thidiazuron, 1-5% sucrose, and 0.1-1% agar.

[0016] Optionally, the absorption surface induction medium comprises the following components: MS medium, 1.0-1.5 mg / L thidiazuron, 3% sucrose, and 0.7% agar.

[0017] The present application also provides a method for in vitro rapid propagation of D. bulbifera var. liliifolium, wherein the subculture step is replaced by: inoculating the stem segments with buds of D. bulbifera var. liliifolium into an absorption surface induction medium for absorption surface induction, wherein the absorption surface induction medium comprises MS medium and thidiazuron.

[0018] Preferably, the concentration of thidiazuron in the absorption surface induction medium is 1.0-1.5 mg / L.

[0019] The present application has found that the absorption surface can be expanded more quickly in the MS medium containing 6-benzylaminopurine, and therefore:

[0020] Preferably, the stem segments subjected to absorption surface induction are inoculated into an absorption surface expansion medium for absorption surface expansion culture, wherein the absorption surface expansion medium comprises MS medium and 6-benzylaminopurine.

[0021] Preferably, the concentration of 6-benzylaminopurine in the absorption surface expansion medium is 3-7 mg / L.

[0022] That is, the method for in vitro rapid propagation of D. bulbifera var. liliifolium comprises the following steps:

[0023] (1) disinfecting the stem segments (explant) with buds of D. bulbifera var. liliifolium and performing primary culture;

[0024] (2) inoculating the stem segments subjected to primary culture in step (1) into an absorption surface induction medium for absorption surface induction;

[0025] (3) inoculating the stem segments subjected to absorption surface induction in step (2) into an absorption surface expansion medium for absorption surface expansion culture;

[0026] (4) taking the expanded absorbing surface formed in step (3) to carry out adventitious bud induction culture and elongation culture, and obtaining adventitious bud clusters;

[0027] (5) taking the adventitious bud clusters in step (4) to carry out rooting culture, and obtaining Dioscorea composita tissue culture seedlings.

[0028] Specifically, the absorbing surface is a spheroid structure expanded at the axillary bud where the stem segment (explant) contacts with the culture medium, which has the effect of expanding the plant nutrient absorbing area.

[0029] As an alternative embodiment, the adventitious bud induction culture and elongation culture method in the above method is to inoculate the absorbing surface into the adventitious bud induction culture medium and the adventitious bud elongation culture medium for culture; and the rooting culture is to inoculate the adventitious bud clusters into the rooting culture medium for culture.

[0030] As an alternative embodiment, the adventitious bud induction culture medium component includes MS culture medium, 6-benzylaminopurine, naphthalene acetic acid, sucrose and agar; and the adventitious bud elongation culture medium component includes MS culture medium, 6-benzylaminopurine, naphthalene acetic acid, activated carbon, sucrose and agar.

[0031] Alternatively, the adventitious bud induction culture medium component includes MS culture medium, 0.5 mg / L 6-benzylaminopurine, 0.05 mg / L naphthalene acetic acid, 1-5% sucrose (preferably 3%) and 0.1-1% agar (preferably 0.7%).

[0032] Alternatively, the adventitious bud induction culture medium component includes MS culture medium, 1 mg / L 6-benzylaminopurine, 0.1 mg / L naphthalene acetic acid, 1-5% sucrose (preferably 3%) and 0.1-1% agar (preferably 0.7%).

[0033] Alternatively, the adventitious bud elongation culture medium component includes MS culture medium, 0.5 mg / L 6-benzylaminopurine, 0.05 mg / L naphthalene acetic acid, 0.1-1 mg / L activated carbon (preferably 0.7 mg / L), 1-5% sucrose (preferably 3%) and 0.1-1% agar (preferably 0.7%).

[0034] Alternatively, the adventitious bud elongation culture medium component includes MS culture medium, 1 mg / L 6-benzylaminopurine, 0.05 mg / L naphthalene acetic acid, 0.1-1 mg / L activated carbon (preferably 0.7 mg / L), 1-5% sucrose (preferably 3%) and 0.1-1% agar (preferably 0.7%).

[0035] Optionally, the adventitious shoot elongation medium components include 1 / 2MS medium, 1 mg / L 6-benzylaminopurine, 0.1 mg / L naphthalene acetic acid, 0.1-1 mg / L activated charcoal (preferably 0.7 mg / L), 1-5% sucrose (preferably 3%), and 0.1-1% agar (preferably 0.7%).

[0036] As an alternative embodiment, the rooting medium components include 1 / 2MS medium, naphthalene acetic acid, activated charcoal, sucrose, and agar.

[0037] Optionally, the rooting medium components include 1 / 2MS medium, 0.1 mg / L naphthalene acetic acid, 0.1-1 mg / L activated charcoal (preferably 0.7 mg / L), 1-5% sucrose (preferably 3%), and 0.1-1% agar (preferably 0.7%).

[0038] Optionally, the rooting medium components include 1 / 2MS medium, 0.2 mg / L naphthalene acetic acid, 0.1-1 mg / L activated charcoal (preferably 0.7 mg / L), 1-5% sucrose (preferably 3%), and 0.1-1% agar (preferably 0.7%).

[0039] Optionally, the rooting medium components include 1 / 2MS medium, 0.2 mg / L naphthalene acetic acid, 0.1-1 mg / L activated charcoal (preferably 0.7 mg / L), 1-5% sucrose (preferably 3%), and 0.1-1% agar (preferably 0.7%).

[0040] In particular, the MS medium includes the following components: ammonium nitrate, potassium nitrate, calcium chloride, magnesium sulfate, potassium dihydrogen phosphate, manganese sulfate, zinc sulfate, cobalt chloride, copper sulfate, boric acid, sodium molybdate, potassium iodide, ferrous sulfate, disodium ethylenediaminetetraacetate, nicotinic acid, vitamin B6, vitamin Bl, myo-inositol, and glycine.

[0041] More specifically, the MS medium comprises the following ingredients: ammonium nitrate 1550-1750 mg / L, potassium nitrate (KNO3) 1800-2000 mg / L, calcium chloride (CaCl2.2H2O) 400-480 mg / L, magnesium sulfate (MgSO4.7H2O) 350-400 mg / L, potassium dihydrogen phosphate (KH2PO4) 150-200 mg / L, manganese sulfate (MnSO4.4H2O) 15-30 mg / L, zinc sulfate (ZnSO4.7H2O) 5-10 mg / L, cobalt chloride (CoCl2.6H2O) 0.01-0.05 mg / L, copper sulfate (CuSO4.5H2O) 0.01-0.05 mg / L, boric acid (H3BO3) 5-10 mg / L, sodium molybdate (Na2MoO4.2H2O) 0.1-0.5 mg / L, potassium iodide (KI) 0.5-1 mg / L, ferrous sulfate (FeSO4.7H2O) 20-30 mg / L, disodium ethylenediaminetetraacetate (Na2-EDTA.2H2O) 35-40 mg / L, nicotinic acid 0.1-1 mg / L, Vitamin B6 0.1-1 mg / L, Vitamin B1 0.05-0.15 mg / L, inositol 80-120 mg / L, glycine 1-3 mg / L.

[0042] Specifically, the 1 / 2MS medium comprises the following ingredients: potassium nitrate, ammonium nitrate, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, potassium iodide, manganese sulfate, zinc sulfate, potassium dihydrogen phosphate, sodium molybdate, copper sulfate, cobalt chloride, ferrous sulfate, boric acid, disodium ethylenediaminetetraacetate, inositol, glycine, nicotinic acid, pyridoxine hydrochloride, thiamine hydrochloride.

[0043] More specifically, the 1 / 2MS medium comprises the following ingredients: potassium nitrate (KNO3) 900-1000 mg / L, ammonium nitrate (NH4NO3) 500-1000 mg / L, potassium dihydrogen phosphate (KH2PO4) 50-100 mg / L, magnesium sulfate (MgSO4·7H2O) 50-150 mg / L, calcium chloride (CaCl2·2H2O) 200-250 mg / L, potassium iodide (KI) 0.5-1 mg / L, manganese sulfate (MnSO4·4H2O) 10-20 mg / L, zinc sulfate (ZnSO4·7H2O) 5-10 mg / L, potassium dihydrogen phosphate (KH2PO4) 50-100 mg / L, sodium molybdate (Na2MoO4·2H2O) 0.1-0.5 mg / L, copper sulfate (CuSO4·5H2O) 0.01-0.05 mg / L, cobalt chloride (CoCl2·6H2O) 0.01-0.05 mg / L, ferrous sulfate (FeSO4·7H2O) 20-30 mg / L, boric acid (H3BO3) 5-10 mg / L, disodium ethylenediaminetetraacetate (Na2-EDTA·2H2O) 35-40 mg / L, inositol 80-120 mg / L, glycine 1-3 mg / L, nicotinic acid 0.1-1 mg / L, pyridoxine HCl 0.1-1 mg / L, thiamine HCl 0.05-0.15 mg / L.

[0044] As an alternative embodiment, the method for in vitro rapid propagation of D. japonica specifically comprises the following steps:

[0045] (1) Explant disinfection and primary culture: selecting D. japonica seedling semi-lignified stem segments, preparing bud-bearing stem segments, washing and disinfecting the bud-bearing stem segments, and then inoculating them into primary culture medium for culture;

[0046] (2) Absorption surface induction and expansion culture: placing the sterile bud-bearing stem segments obtained in step (1) horizontally in absorption surface induction medium for culture to induce the formation of absorption surfaces, and then transferring the bud-bearing stem segments with induced absorption surfaces to absorption surface expansion medium to induce absorption surface expansion, and obtaining bud-bearing stem segments with expanded absorption surfaces;

[0047] (3) Adventitious bud induction and elongation culture: cutting the expanded absorption surfaces induced in step (2) from the stem segments, transferring them to adventitious bud induction medium for culture, and then transferring them to adventitious bud elongation medium for culture, and obtaining adventitious bud clusters;

[0048] (4) Rooting culture: transferring the adventitious bud clusters obtained in step (3) to rooting culture medium for rooting culture, and obtaining D. japonica tissue culture seedlings.

[0049] Specifically, the disinfection method in step (1) is as follows: disinfecting the stem segments with buds with 75% alcohol for 30s, then washing with sterile water for 3-5 times, and then disinfecting the stem segments with buds with 1% sodium hypochlorite for 2-5 min.

[0050] Specifically, the primary culture medium comprises the following components: MS medium, 1.0 mg / L 6-benzylaminopurine, 0.1 mg / L naphthalene acetic acid, 0.1 mg / L GA3, 3% sucrose, and 0.7% agar.

[0051] Specifically, the culture conditions in steps (1)-(4) are as follows: the culture temperature is 23±2℃, the light condition is 1500-2500lx, and the light duration is 16 hours per day.

[0052] Specifically, the culture in step (1) is dark culture for 3 days followed by light culture, and the light culture condition is 1500-2500lx, 16 hours per day.

[0053] As an alternative embodiment, the tissue culture seedlings obtained through steps (1)-(4) are hardened and transplanted, and the hardening and transplanting method is as follows: the tissue culture seedlings are cultured with the cover removed for 3-5 days, the tissue culture seedlings are divided into single seedlings with roots, stems and leaves before being transplanted, and the single seedlings are transplanted into the substrate and hardened under the conditions of a temperature of 23-25℃ and a humidity of 75-85%.

[0054] As an alternative embodiment, the substrate is 2 / 3 nutrient soil and 1 / 3 vermiculite.

[0055] The present application has the following beneficial effects:

[0056] 1. The present application develops a kind of in vitro rapid propagation method of D. japonica by using tissue culture technology. The present application uses stem segments with buds of D. japonica as explants to induce the formation of swollen absorption surface, and then induces adventitious buds on the basis of the swollen absorption surface. The formed adventitious bud clusters are subjected to rooting culture, and finally the tissue culture seedlings with roots, stems and leaves are obtained. By inducing the formation of swollen absorption surface in the explants, the adaptability of the explants to the tissue culture environment is enhanced, the vitrification and browning phenomena of the tissue culture seedlings of D. japonica in the cultivation process are overcome, the survival rate of the tissue culture seedlings is improved, the growth of the stable tissue culture seedlings is not required to be subcultured multiple times, the proliferation and expansion speed of the tissue culture seedlings is improved, and the culture time of the tissue culture seedlings is shortened.

[0057] 2. The tissue culture seedlings produced by the in vitro rapid propagation method of the dioscorea composita have unified genotype, small variation probability, and are easy to standardize, and can be applied to the stable production of dioscorea complita high-quality seedlings. The application solves the problems of different seedling qualities caused by the large offspring separation and unstable traits of traditional seed propagation, the high cost and low propagation coefficient of traditional rhizome propagation, and overcomes the problem that the traditional propagation method cannot be put into large-scale industrial development. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 Figures for the growth stages of the in vitro rapid propagation system (a is the growth situation diagram of the dioscorea composita stem segment explant with buds; b is the growth situation diagram of the primary culture of the explant; c is the growth situation diagram of the absorption surface induction culture of the stem segment with buds; d is the absorption surface induction situation diagram of the stem segment with buds; e is the growth situation diagram of the stem segment with buds for absorption surface expansion culture; f is the growth situation diagram of the adventitious bud induction for 30 days; g and h are the growth situation diagrams of the elongation culture of the adventitious bud; i is the growth situation diagram of the rooting culture).

[0059] Figure 2 Figures for the growth situation of the seedlings (a is the growth situation diagram of the tissue culture seedling; b is the growth situation diagram of the root system of the seedling; c is the growth situation diagram of the seedling before transplanting).

[0060] Figure 3 Figure for the phenotype of the bud seedling when vitrification occurs in Comparative Example 1.

[0061] Figure 4 Figure for the phenotype of the bud seedling when browning occurs in Comparative Example 1. DETAILED DESCRIPTION

[0062] The present application will be further described in conjunction with the drawings and specific examples in the specification, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and devices used in the present application are conventional reagents, methods and devices in the technical field.

[0063] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0064] MS medium components: ammonium nitrate (NH4NO3) 1650 mg / L, potassium nitrate (KNO3) 1900 mg / L, calcium chloride (CaCl2·2H2O) 440 mg / L, magnesium sulfate (MgSO4·7H2O) 370 mg / L, potassium dihydrogen phosphate (KH2PO4) 170 mg / L, manganese sulfate (MnSO4·4H2O) 22.3 mg / L, zinc sulfate (ZnSO4·7H2O) 8.6 mg / L, cobalt chloride (CoCl2·6H2O) 0.025 mg / L, copper sulfate (CuSO4·5H2O) 0.025 mg / L, boric acid (H3BO3) 6.2 mg / L, sodium molybdate (Na2MoO4·2H2O) 0.25 mg / L, potassium iodide (KI) 0.83 mg / L, ferrous sulfate (FeSO4·7H2O) 27.8 mg / L, disodium ethylenediaminetetraacetate (Na2-EDTA·2H2O) 37.2 mg / L, nicotinic acid 0.5 mg / L, Vitamin B6 0.5 mg / L, Vitamin B1 0.1 mg / L, inositol 100 mg / L, glycine 2 mg / L.

[0065] 1 / 2MS medium components: potassium nitrate (KNO3) 950 mg / L, ammonium nitrate (NH4NO3) 825 mg / L, potassium dihydrogen phosphate (KH2PO4) 85 mg / L, magnesium sulfate (MgSO4·7H2O) 90.35 mg / L, calcium chloride (CaCl2·2H2O) 220 mg / L, potassium iodide (KI) 0.83 mg / L, manganese sulfate (MnSO4·4H2O) 16.9 mg / L, zinc sulfate (ZnSO4·7H2O) 8.6 mg / L, potassium dihydrogen phosphate (KH2PO4) 85 mg / L, sodium molybdate (Na2MoO4·2H2O) 0.25 mg / L, copper sulfate (CuSO4·5H2O) 0.025 mg / L, cobalt chloride (CoCl2·6H2O) 0.025 mg / L, ferrous sulfate (FeSO4·7H2O) 27.8 mg / L, boric acid (H3BO3) 6.2 mg / L, disodium ethylenediaminetetraacetate (Na2-EDTA·2H2O) 36.7 mg / L, inositol 100 mg / L, glycine 2 mg / L, nicotinic acid 0.5 mg / L, pyridoxine HCl 0.5 mg / L, thiamine HCl 0.1 mg / L.

[0066] Primary culture medium (pH = 5.8-6.0): MS medium + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 0.1 mg / L GA3 + 3% sucrose + 0.7% agar.

[0067] Subculture medium: MS medium + 1.5 mg / L 6-BA + 0.1 mg / L NAA + 3% sucrose + 0.7% agar.

[0068] Thiabenzomerazole (TDZ), CAS No.: 51707-55-2, Structural Formula:

[0069] 6-Benzylaminopurine (6-BA), CAS No.: 1214-39-7, structural formula is

[0070] Naphthaleneacetic acid (NAA), CAS No.: 86-87-3, structural formula is

[0071] Example 1: Establishment of a sterile culture system

[0072] Select robust, semi-lignified stem segments from seedlings of Dioscorea opposita. Figure 1 (See Figure a). Remove the leaves and cut the stem segments into 1-2cm sections with buds, one bud per segment. Soak and stir in detergent solution, gently brush the axillary buds of the stem segments with a soft brush, and rinse with running water for 15 minutes to complete the initial disinfection of the explants. Then place them in a clean bench and disinfect by soaking in 75% alcohol for 30 seconds, rinsing with sterile water 3-5 times, and then by soaking in 1% sodium hypochlorite for 3 minutes, rinsing with sterile water 3-5 times. Inoculate the disinfected explants into primary culture medium for 3 days of dark culture. After that, transfer them to a primary culture environment with a light intensity of 1500-2500 lx, a photoperiod of 16 h / d, and a temperature of 23℃. After 30 days of culture, sterile seedlings are obtained. Figure 1 (Figure b).

[0073] Example 2: Induction and Expansion Culture of Absorbent Surface

[0074] I. Experimental Methods

[0075] The aseptic seedlings obtained in Example 1 were cut into 1cm aseptic stem segments with buds, with one bud per node. These aseptic stem segments with buds were horizontally inoculated onto absorbance induction medium containing different concentrations of TDZ (0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg / L) for absorbance induction culture. Figure 1 (See Figure c) to compare the effects of different concentrations of TDZ on the absorption surface induction effect of budded stem segments, and to determine the optimal absorption surface induction medium.

[0076] Then the expanded absorption surface was inoculated into the absorption surface expansion medium with different concentrations of 6-BA (1, 3, 5, 7, 9 mg / L) to expand the absorption surface, and the effects of different concentrations of 6-BA on the absorption surface expansion were compared to determine the most suitable absorption surface expansion medium.

[0077] The culture conditions of the absorption surface induction culture and the absorption surface expansion culture were as follows: the culture temperature was 23±2℃, the light condition was 1500-2500 lx, and the light duration was 16 hours per day.

[0078] The absorption surface induction medium was composed of MS medium + TDZ (0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg / L) + 3% sucrose + 0.7% agar, and the pH value was 5.8-6.0.

[0079] The absorption surface expansion medium was composed of MS medium + 6-BA (1, 3, 5, 7, 9 mg / L) + 3% sucrose + 0.7% agar, and the pH value was 5.8-6.0.

[0080] The absorption surface induction rate (%) = (the number of the explants with the induced absorption surface / the total number of the inoculated explants) * 100

[0081] The absorption surface expansion rate (%) = (the number of the explants with the expanded absorption surface / the total number of the inoculated explants) * 100

[0082] II. Experimental results

[0083] The effects of different concentrations of TDZ on the absorption surface induction of the stem segments with buds are shown in Table 1. As shown in Table 1, with the increase of the concentration of TDZ, the induction rate of the absorption surface showed a trend of first increasing and then decreasing. When the concentration of TDZ was 1.5 mg / L, the induction effect on the absorption surface of the axillary bud was the best, and the low and high concentrations of TDZ could make the axillary bud germinate, which significantly reduced the induction rate of the absorption surface (as shown in the d graph of FIG. 1). Figure 1 During the experiment, it was found that the stem segments with buds inoculated into the absorption surface induction medium after the primary culture could form the absorption surface after about 21 days of culture.

[0084] The effects of different concentrations of 6-BA on the absorption surface expansion culture are shown in Table 2. As shown in Table 2, with the increase of the concentration of 6-BA, the expansion rate of the absorption surface showed a trend of first increasing and then decreasing. When the concentration of 6-BA was 3-7 mg / L, the absorption surface could be expanded. When the concentration of 6-BA was 5 mg / L, the expansion efficiency of the absorption surface was the highest, which was 77.79%, and the growth state was the best, which was the green expanded ball. Therefore, the suitable concentration of 6-BA for the expansion and development culture of the absorption surface was 5 mg / L (as shown in the e graph of FIG. 2). Figure 1 The absorption surface could be cultured for 20-30 days in the absorption surface expansion medium.

[0085] Table 1 Effect of different concentrations of TDZ on the induction of the absorption surface of stem segments with buds

[0086]

[0087] Table 2 Effect of different concentrations of 6-BA on the swelling culture of the absorption surface

[0088]

[0089] Example 3 Induction and elongation culture of adventitious buds

[0090] I. Experimental method

[0091] The stem segments with good swelling of the absorption surface after the culture in Example 2 were transferred to an adventitious bud induction medium with different combinations of 6-BA (0.5, 1.0, 1.5 mg / L) and NAA (0.05, 0.1, 0.2 mg / L) for the induction and culture of adventitious buds (the culture time was 15-25 days), and the effects of different combinations of hormones on the induction of adventitious buds were compared to determine the optimum adventitious bud induction medium.

[0092] Subsequently, the adventitious bud clusters were transferred to an adventitious bud elongation medium with different concentrations of 6-BA (0.5, 1.0, 1.5 mg / L) and NAA (0.05, 0.1, 0.2 mg / L) for the elongation culture of adventitious buds (the culture time was 20-30 days), and the effects of different combinations of hormones on the elongation of adventitious buds were compared to determine the optimum adventitious bud elongation medium.

[0093] The components of the adventitious bud induction medium were MS medium + 6-BA (0.5, 1.0, 1.5 mg / L) + NAA (0.05, 0.1, 0.2 mg / L) + 3% sucrose + 0.7% agar.

[0094] The components of the adventitious bud elongation medium were MS medium + 6-BA (0.5, 1.0, 1.5 mg / L) + NAA (0.05, 0.1, 0.2 mg / L) + 0.7 mg / L AC + 3% sucrose + 0.7% agar.

[0095] II. Experimental results

[0096] As shown in Table 3, a combination of a lower concentration of 6-BA and NAA is more suitable for the induction and culture of adventitious buds. When 1.0 mg / L of 6-BA and 0.1 mg / L of NAA were added, the induction effect of adventitious buds was the best, with an induction rate of 73.33% and an average number of adventitious buds of 6.67, and the adventitious buds grew healthily (see Figures f-g). Figure 1

[0097] ​From Table 4, it can be seen that when the hormone combination is 0.5 mg / L 6-BA and 0.05 mg / L NAA, the elongation culture of adventitious buds is best, and the plant growth is vigorous, with an average seedling height of 4.03 cm. It can also be seen that when the ratio of cytokinin to auxin in the hormone combination is 10:1, the average seedling height is higher than that of other hormone combinations (h graph). Figure 1

[0098] Table 3 Influence of different ratios of plant hormone combinations on induction of adventitious buds

[0099]

[0100] Table 4 Influence of different ratios of plant hormone combinations on elongation of adventitious buds

[0101]

[0102]

[0103] Example 4 Rooting culture

[0104] I. Experimental method

[0105] The well-grown adventitious bud seedlings of Example 3 were transferred to different formulations of rooting induction medium for rooting culture (rooting culture time was 25-30 days), the effects of different treatments on root induction of bud seedlings were compared, and the most suitable rooting induction medium was determined.

[0106] Six different formulations of rooting induction medium were set as different treatments;

[0107] The rooting induction medium of treatment 1 had the following formulation: 1 / 2MS medium + 0.1 mg / L NAA + 0.7 mg / L AC + 3% sucrose + 0.7% agar;

[0108] The rooting induction medium of treatment 2 had the following formulation: 1 / 2MS medium + 0.2 mg / L NAA + 0.7 mg / L AC + 3% sucrose + 0.7% agar;

[0109] The rooting induction medium of treatment 3 had the following formulation: 1 / 2MS medium + 0.3 mg / L NAA + 0.7 mg / L AC + 3% sucrose + 0.7% agar;

[0110] The rooting induction medium of treatment 4 had the following formulation: MS medium + 0.1 mg / L NAA + 0.7 mg / L AC + 3% sucrose + 0.7% agar;

[0111] The rooting induction medium of treatment 5 had the following formulation: MS medium + 0.2 mg / L NAA + 0.7 mg / L AC + 3% sucrose + 0.7% agar; ​

[0112] The rooting induction medium formula of the treatment 6 is: MS medium + 0.3 mg / L NAA + 0.7 mg / L AC + 3% sucrose + 0.7% agar.

[0113] II. Experimental results

[0114] As shown in Table 5, the induction rate of adventitious roots in the 1 / 2MS medium is generally higher than that in the MS medium, so the 1 / 2MS medium is more conducive to the induction culture of adventitious roots of the bud seedlings. Different concentrations of NAA can induce adventitious roots, and with the increase of the NAA concentration, the rooting rate of the bud seedlings shows a trend of first increasing and then decreasing. When the NAA concentration is 0.2 mg / L, the rooting rate of the bud seedlings is the highest, reaching 86.67%, and the induced adventitious roots are relatively thick and have more lateral roots (see FIG. 1). Figure 1

[0115] Table 5 Influence of rooting induction medium of different treatments on root induction of bud seedlings

[0116]

[0117]

[0118] Example 5 Raising seedlings and transplanting

[0119] Example 4 Growth of seedlings of the tissue culture seedlings after rooting induction Figure 2 As shown in FIG. 2a, the growth of the roots of the seedlings is shown in FIG. 2b, and the growth of the seedlings before transplanting is shown in FIG. 2c. Figure 2 Figure 2 As shown in FIG. 2a, the growth of the roots of the seedlings is shown in FIG. 2b, and the growth of the seedlings before transplanting is shown in FIG. 2c.

[0120] The tissue culture seedlings of the Anemarrhena asphodeloides obtained by rooting induction in Example 4 were cultured in the culture room for 3-5 days, and before transplanting, the tissue culture seedlings were divided into single plants with roots, stems and leaves, and the single seedlings were transferred to the transplanting culture container with a transparent plastic cover, and were raised in the culture box at a temperature of 23-25°C and a humidity of 75-85%. The survival of the transplanted seedlings indicates that the transplanted seedlings can grow and sprout normally.

[0121] Comparative Example 1

[0122] The sterile bud seedlings obtained in Example 1 were cut into 1 cm sterile stem segments with buds, and the sterile stem segments with buds were inoculated into the subculture medium for subculture (without absorption surface induction), and the subculture was performed every 30 d.

[0123] ​​The results of subculture of the aseptic stem segments with buds are shown in Table 6. As shown in Table 6, with the increase of subculture times, the average bud number and the average plant height are increased, and the average bud number and the average plant height of the bud seedlings of the third subculture are the highest. In the first subculture, most of the bud seedlings change from dark green to light green, and the growth of the bud seedlings is poor, and the seedlings are thin and weak. After the second subculture, the thin and weak bud seedlings stop growing and gradually become white, that is, the bud seedlings appear vitrification phenomenon Figure 3 ), and part of the bud seedlings with good growth also gradually appear different degrees of browning Figure 4 ), and the growth is weakened. After the third subculture, the bud seedlings tend to be stable, but the proliferation coefficient is slow. In the process of subculture, part of the bud seedlings cannot adapt to the tissue culture environment, and the growth is poor or even stops, resulting in poor proliferation effect. The bud seedlings need to be subcultured continuously to grow stably. The experimental data show that at least 3 times of subculture, the plant can grow normally.

[0124] Table 6 Influence of subculture times on the bud seedlings of subculture

[0125]

[0126] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations and simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and all are included in the protection scope of the present application.

Claims

1. A method for in vitro rapid propagation of Dioscorea composita, characterized by, It comprises the following steps: (1) disinfecting the stem section with buds of Dioscorea composita and then carrying out primary culture; (2) inoculating the stem section with buds of Dioscorea composita after primary culture in step (1) into an absorption surface induction medium to carry out absorption surface induction, wherein the absorption surface induction medium comprises the following components: MS medium, 1.0-1.5 mg / L thidiazuron, 1-5% sucrose and 0.1-1% agar; (3) inoculating the stem section with buds after absorption surface induction in step (2) into an absorption surface expansion medium to carry out absorption surface expansion culture, wherein the absorption surface expansion medium comprises the following components: MS medium, 3-7 mg / L 6-benzylaminopurine, 1-5% sucrose and 0.1-1% agar; (4) taking the expanded absorption surface formed in step (3) to carry out adventitious bud induction culture and elongation culture, and obtaining an adventitious bud cluster; the medium components for the adventitious bud induction culture comprise MS medium, 0.5-1 mg / L 6-benzylaminopurine, 0.05-0.1 mg / L naphthaleneacetic acid, 1-5% sucrose and 0.1-1% agar; the medium components for the adventitious bud elongation culture comprise MS medium, 0.5-1 mg / L 6-benzylaminopurine, 0.05-0.1 mg / L naphthaleneacetic acid, 0.1-1 mg / L activated carbon, 1-5% sucrose and 0.1-1% agar; (5) taking the adventitious bud cluster in step (4) to carry out rooting culture, and obtaining Dioscorea composita tissue culture seedlings; the medium components for the rooting culture comprise 1 / 2MS medium, 0.1-0.2 mg / L naphthaleneacetic acid, 0.1-1 mg / L activated carbon, 1-5% sucrose and 0.1-1% agar.

2. The method of claim 1, wherein, The MS medium comprises the following components: ammonium nitrate, potassium nitrate, calcium chloride, magnesium sulfate, potassium dihydrogen phosphate, manganese sulfate, zinc sulfate, cobalt chloride, copper sulfate, boric acid, sodium molybdate, potassium iodide, ferrous sulfate, disodium ethylenediaminetetraacetate, nicotinic acid, vitamin B6, vitamin B1, myo-inositol and glycine.

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