Efficient in-vitro rapid propagation method for dioscorea composita

Abstract

The invention belongs to the technical field of medicinal plant tissue culture. More specifically, the invention provides an in-vitro rapid propagation method of Dioscorea composita, stem segments with buds of Dioscorea composita are inoculated to an absorption surface induction culture medium for absorption surface induction, and the absorption surface induction culture medium comprises an MS culture medium and thidiazuron (TDZ). According to the method disclosed by the invention, the stem segments with buds of the dioscorea composita are used as the explants to induce the stem segments to generate expanded absorption surfaces, so that the adaptability of the explants to a tissue culture environment is enhanced, vitrification and browning phenomena of tissue culture seedlings of the dioscorea composita in a culture process are overcome, the growth vigor of the tissue culture seedlings is stabilized without multiple subculture, the proliferation and propagation speed of the tissue culture seedlings is increased, and the survival rate of the tissue culture seedlings is increased. The culture time of tissue culture seedlings is shortened. In addition, the tissue culture seedlings produced by the rapid in-vitro propagation method of the dioscorea composita are uniform in genotype, small in variation probability and easy to standardize in the propagation process, can be applied to stable production of high-quality seedlings of the dioscorea composita, and have good application prospects and value.

Description

Technical Field

[0001] The invention belongs to the technical field of medicinal plant tissue culture, and more specifically, relates to a high-efficiency in vitro rapid propagation method of Dioscorea zingiberensis. Background Art

[0002] Dioscorea composita Hemsl., also known as Mexican yam, belongs to the Dioscorea genus of the Dioscoreaceae family. It is a perennial twining herb, which is mainly distributed in tropical and subtropical regions of Asia and America. It has multiple economic values, such as being used to extract diosgenin and other diosgenin saponins. Diosgenin, commonly known as saponin, is a natural secondary metabolite with multiple pharmacological effects. It can be used to prevent and treat cancer, asthma, diabetes, arthritis, cardiovascular disease, hypertension, coronary heart disease, vascular sclerosis and other diseases; it is an important raw material for synthesizing more than 300 steroid hormone drugs such as adrenal cortex hormones and sex hormones, and is known as "medicinal gold". Moreover, most of the corticosteroids, sex hormones and anabolic hormones produced in various countries around the world use diosgenin as the basic raw material, which has extremely high economic and medicinal value, so the demand for diosgenin is very large. Therefore, it is of great significance to efficiently and massively propagate Dioscorea zingiberensis to meet the future market and development needs of diosgenin.

[0003] At present, the conventional cultivation methods of Dioscorea zingiberensis mainly include seed propagation and rhizome propagation. Although seed propagation has the advantages of relatively simple operation and low cost, the seed storage and germination effects are greatly affected by natural conditions such as season, climate, cultivation environment, and pests and diseases. In addition, there is still a phenomenon of difficulty in seed setting during the seed propagation of Dioscorea zingiberensis in my country, which seriously limits its propagation rate and has certain limitations in its application in large-scale production. Rhizome propagation is limited by the long growth cycle of Dioscorea zingiberensis, low underground rhizome propagation coefficient, high cost, and large seed usage, making it difficult to quickly promote planting. As a result, there is a mismatch between the existing Dioscorea zingiberensis seedling propagation speed and market demand, which affects the development of the Dioscorea zingiberensis industry.

[0004] Plant tissue culture rapid propagation is an important technical means commonly used in the process of seed propagation, and is now widely used in the production fields of fruits and vegetables, garden trees, forests, economic crops and medicinal plants. The application of plant tissue culture rapid propagation has significantly improved the efficiency of plant seedling production, increased the yield of plant seedlings, promoted the factory production of plant seedlings, and brought huge economic and social benefits. However, the current tissue culture rapid propagation of Dioscorea zingiberensis has the problem that it requires multiple subcultures to grow the tissue culture seedlings stably. At the same time, the tissue culture seedlings are prone to vitrification or browning during multiple subcultures. That is, after the explants of Dioscorea zingiberensis are disinfected and cultured initially, multiple subcultures are required to continue the next step of adventitious bud induction, elongation culture and rooting culture, resulting in low efficiency and instability in the tissue culture propagation of Dioscorea zingiberensis.

[0005] Therefore, researching and developing efficient in vitro rapid propagation technology of Dioscorea zingiberensis is of great significance for promoting the factory-scale production of Dioscorea zingiberensis tissue culture rapid propagation seedlings. Summary of the invention

[0006] The invention provides a method for rapid in vitro propagation of Dioscorea zingiberensis in order to solve the problems of vitrification or browning that the existing Dioscorea zingiberensis tissue culture seedlings are prone to and the problem that the growth of the tissue culture seedlings can be stable only after multiple subcultures.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] After extensive research and exploration, the present invention has developed a culture medium capable of inducing the stem segments (explants) with buds of Dioscorea zingiberensis to form an enlarged spherical structure (named as the absorption surface) at the axillary buds in contact with the culture medium, which is named as the absorption surface induction culture medium and the absorption surface enlargement culture medium. After the stem segments (explants) with buds of Dioscorea zingiberensis are disinfected and cultured initially, subculture is no longer required, and the absorption surface induction culture medium and the absorption surface enlargement culture medium are used for culture instead, and then the next step of adventitious bud induction, elongation culture and rooting culture is directly carried out. This technology not only omits the steps of multiple subcultures and shortens the tissue culture time, but also overcomes the phenomenon of vitrification and browning of Dioscorea zingiberensis tissue culture seedlings during the cultivation process.

[0009] Therefore, the present invention provides the use of an MS culture medium containing thidiazuron and an MS culture medium containing 6-benzylaminopurine in in vitro rapid propagation of Dioscorea zingiberensis.

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

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

[0012] As an optional embodiment, the absorption surface induction medium also includes 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 expansion medium comprises the following components: MS medium, 3-7 mg / L 6-benzylaminopurine, 1-5% sucrose and 0.1-1% agar.

[0016] Optionally, the absorption surface expansion medium comprises the following components: MS medium, 3-7 mg / L 6-benzylaminopurine, 3% sucrose and 0.7% agar.

[0017] The present invention also provides a method for in vitro rapid propagation of Dioscorea zingiberensis, which replaces the subculture step with: inoculating the stem segments with buds of Dioscorea zingiberensis into an absorption surface induction culture medium for absorption surface induction, wherein the absorption surface induction culture medium comprises an MS culture 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 invention has found that the absorption surface expands faster in the MS medium containing 6-benzylaminopurine, so:

[0020] Preferably, the stem segments induced by the absorption surface are inoculated into an absorption surface expansion culture medium for absorption surface expansion culture, wherein the absorption surface expansion culture 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 in vitro rapid propagation method of Dioscorea zingiberensis comprises the following steps:

[0023] (1) sterilizing the stem segments with buds (explants) of Dioscorea zingiberensis and performing primary culture;

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

[0025] (3) inoculating the stem segments induced by the absorption surface in step (2) into the absorption surface expansion culture medium for absorption surface expansion culture;

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

[0027] (5) taking the adventitious bud clusters of step (4) for rooting culture to obtain Dioscorea zingiberensis tissue culture seedlings.

[0028] Specifically, the absorption surface is an enlarged spherical structure at the axillary bud where the stem segment (explant) contacts the culture medium, and the structure has the function of expanding the nutrient absorption area of ​​the plant.

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

[0030] As an optional embodiment, the above-mentioned adventitious bud induction medium components include MS medium, 6-benzylaminopurine, naphthaleneacetic acid, sucrose and agar; the adventitious bud elongation medium components include MS medium, 6-benzylaminopurine, naphthaleneacetic acid, activated carbon, sucrose and agar.

[0031] Optionally, the adventitious bud induction medium components include MS medium, 0.5 mg / L 6-benzylaminopurine, 0.05 mg / L naphthaleneacetic acid, 1-5% sucrose (preferably 3%) and 0.1-1% agar (preferably 0.7%).

[0032] Optionally, the adventitious bud induction medium components include MS medium, 1 mg / L 6-benzylaminopurine, 0.1 mg / L naphthaleneacetic acid, 1-5% sucrose (preferably 3%) and 0.1-1% agar (preferably 0.7%).

[0033] Optionally, the adventitious shoot elongation medium components include MS medium, 0.5 mg / L 6-benzylaminopurine, 0.05 mg / L naphthaleneacetic 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] Optionally, the adventitious shoot elongation medium components include MS medium, 1 mg / L 6-benzylaminopurine, 0.05 mg / L naphthaleneacetic 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 MS medium, 1 mg / L 6-benzylaminopurine, 0.1 mg / L naphthaleneacetic 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%).

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

[0037] Optionally, the rooting medium components include 1 / 2 MS medium, 0.1 mg / L naphthaleneacetic 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%).

[0038] Optionally, the rooting medium components include 1 / 2 MS medium, 0.2 mg / L naphthaleneacetic 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%).

[0039] Optionally, the rooting medium components include MS medium, 0.2 mg / L naphthaleneacetic 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%).

[0040] Specifically, the MS culture medium includes the following ingredients: 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 ethylenediaminetetraacetic acid, niacin, vitamin B6, vitamin B1, inositol and glycine.

[0041] More specifically, the MS culture medium includes 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) 0.01-0.05mg / L, Copper sulfate (CuSO4·5H2O) 0.01-0.05mg / L, Boric acid (H3BO3) 5-10mg / L, Sodium molybdate (Na2MoO4·2H2O) 0.1-0.5mg / L, Potassium iodide (KI) 0.5-1mg / L, Ferrous sulfate (FeSO4·7H2O) 20-30mg / L, Disodium ethylenediaminetetraacetate (Na2-EDTA·2H2O) 35-40mg / L, Nicotinic acid acid) 0.1-1mg / L, Vitamin B6 (Vitamin B6) 0.1-1mg / L, Vitamin B1 (Vitamin B1) 0.05-0.15mg / L, Inositol (Inositol) 80-120mg / L, Glycine (Glycine) 1-3mg / L.

[0042] Specifically, the 1 / 2MS culture medium includes 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 ethylenediaminetetraacetic acid, inositol, glycine, niacin, pyridoxine hydrochloride, and thiamine hydrochloride.

[0043] More specifically, the 1 / 2MS culture 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) 50-150 mg / L 100mg / L, Sodium molybdate (Na2MoO4·2H2O) 0.1-0.5mg / L, Copper sulfate (CuSO4·5H2O) 0.01-0.05mg / L, Cobalt chloride (CoCl2·6H2O) 0.01-0.05mg / L, Ferrous sulfate (FeSO4·7H2O) 20-30mg / L, Boric acid (H3BO3) 5-10mg / L, Disodium ethylenediaminetetraacetic acid (Na2-EDTA·2H2O) 35-40mg / L, Inositol 80-120mg / L, Glycine 1-3mg / L, Nicotinic acid acid) 0.1-1mg / L, Pyridoxine HCl 0.1-1mg / L, Thiamine HCl 0.05-0.15mg / L.

[0044] As an optional implementation scheme, the in vitro rapid propagation method of Dioscorea zingiberensis comprises the following steps:

[0045] (1) Explant disinfection and primary culture: Select semi-lignified stem segments of Dioscorea zingiberensis seedlings, prepare stem segments with buds, clean and disinfect the stem segments with buds, and then inoculate them into primary culture medium for culture;

[0046] (2) Absorption surface induction and expansion culture: the sterile stem segment with buds obtained in step (1) is horizontally placed in an absorption surface induction culture medium for culture to induce the formation of the absorption surface, and then the stem segment with buds with the absorption surface induced to form is transferred to an absorption surface expansion culture medium to induce the absorption surface to expand, thereby obtaining a stem segment with buds having an expanded absorption surface;

[0047] (3) Adventitious bud induction and elongation culture: the expanded absorption surface induced in step (2) is cut off from the stem segment, transferred to an adventitious bud induction medium for culture, and then transferred to an adventitious bud elongation medium for culture to obtain an adventitious bud cluster;

[0048] (4) Rooting culture: The adventitious bud clusters obtained in step (3) are transferred to a rooting medium for rooting culture to obtain Dioscorea zingiberensis tissue culture seedlings.

[0049] Specifically, the disinfection method in step (1) is: disinfect the stem segments with buds with 75% alcohol for 30 seconds, then wash with sterile water for 3-5 times, and then disinfect the stem segments with buds with 1% sodium hypochlorite for 2-5 minutes.

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

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

[0052] Specifically, the culture in step (1) is performed in dark for 3 days and then in light for 16 hours per day under the conditions of 1500-2500 lx.

[0053] As an optional implementation scheme, the tissue culture seedlings obtained through the above steps (1)-(4) are hardened and transplanted. The specific method of hardening and transplanting is: the tissue culture seedlings are cultured with the cover opened for 3-5 days, and before transplanting, the tissue culture seedlings are divided into individual seedlings with roots, stems and leaves, and the individual seedlings are transferred to a substrate for culture. The hardening and culture are carried out under the conditions of a temperature of 23-25°C and a humidity of 75-85%.

[0054] As an optional embodiment, the matrix is ​​2 / 3 nutrient soil and 1 / 3 vermiculite.

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

[0056] 1. The present invention uses tissue culture technology to develop a method for in vitro rapid propagation of Dioscorea zingiberensis. The present invention uses the stem segment with buds of Dioscorea zingiberensis as an explant to induce it to produce an enlarged absorption surface, and on the basis of the enlarged absorption surface, induces adventitious buds, and culture the formed adventitious bud clusters for rooting, and finally obtains tissue culture seedlings with complete roots, stems and leaves. By inducing the explant to form an enlarged absorption surface, the adaptability of the explant to the tissue culture environment is enhanced, the vitrification and browning phenomena of Dioscorea zingiberensis tissue culture seedlings during the cultivation process are overcome, the survival rate of tissue culture seedlings is improved, and the growth of tissue culture seedlings is stabilized without multiple subcultures, the proliferation and expansion speed of tissue culture seedlings is improved, and the cultivation time of tissue culture seedlings is shortened.

[0057] 2. The tissue culture seedlings produced by the in vitro rapid propagation method of Dioscorea zingiberensis of the present invention have uniform genotypes during the breeding process, low probability of variation, and are easy to standardize, and can be applied to the stable production of high-quality Dioscorea zingiberensis seedlings. The present invention solves the problems of different seedling quality caused by large separation of offspring and unstable traits in traditional seed propagation, high cost and low propagation coefficient of traditional rhizome propagation, and overcomes the problem that traditional propagation methods cannot be put into large-scale factory development. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0059] Figure 2 Figure 1 is a graph showing the growth of mature seedlings (Figure a is a graph showing the growth of mature seedlings from tissue culture; Figure b is a graph showing the growth of the root system of mature seedlings; Figure c is a graph showing the growth of mature seedlings before transplanting).

[0060] Figure 3 This is the phenotype diagram of the seedlings in Comparative Example 1 when vitrification occurs.

[0061] Figure 4 This is the phenotype diagram when the sprouts in Comparative Example 1 turn brown. DETAILED DESCRIPTION

[0062] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

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

[0064] MS medium components: ammonium nitrate (NH4NO3) 1650mg / L, potassium nitrate (KNO3) 1900mg / L, calcium chloride (CaCl2·2H2O) 440mg / L, magnesium sulfate (MgSO4·7H2O) 370mg / L, potassium dihydrogen phosphate (KH2PO4) 170mg / L, manganese sulfate (MnSO4·4H2O) 22.3mg / L, zinc sulfate (ZnSO4·7H2O) 8.6mg / L, cobalt chloride (CoCl2 ·6H2O)0.025mg / L, Copper sulfate (CuSO4·5H2O)0.025mg / L, Boric acid (H3BO3)6.2mg / L, Sodium molybdate (Na2MoO4·2H2O)0.25mg / L, Potassium iodide (KI)0.83mg / L, Ferrous sulfate (FeSO4·7H2O)27.8mg / L, Disodium ethylenediaminetetraacetic acid (Na2-EDTA·2H2O)37.2mg / L, Nicotinic acid0.5mg / L, Vitamin B6(Vitamin B6)0.5mg / L, Vitamin B1(Vitamin B1)0.1mg / L, Inositol(Inositol)100mg / L, Glycine(Glycine)2mg / L.

[0065] 1 / 2MS culture medium components: potassium nitrate (KNO3) 950mg / L, ammonium nitrate (NH4NO3) 825mg / L, potassium dihydrogen phosphate (KH2PO4) 85mg / L, magnesium sulfate (MgSO4·7H2O) 90.35mg / L, calcium chloride (CaCl2·2H2O) 220mg / L, potassium iodide (KI) 0.83mg / L, manganese sulfate (MnSO4·4H2O) 16.9mg / L, zinc sulfate (ZnSO4·7H2O) 8.6mg / L, potassium dihydrogen phosphate (KH2PO4) 85mg / L, molybdenum Sodium sulfate (Na2MoO4·2H2O) 0.25mg / L, copper sulfate (CuSO4·5H2O) 0.025mg / L, cobalt chloride (CoCl2·6H2O) 0.025mg / L, ferrous sulfate (FeSO4·7H2O) 27.8mg / L, boric acid (H3BO3) 6.2mg / L, disodium ethylenediaminetetraacetic acid (Na2-EDTA·2H2O) 36.7mg / L, inositol (Inositol) 100mg / L, glycine (Glycine) 2mg / L, nicotinic acid (Nicotinic acid) 0.5mg / L, pyridoxine hydrochloride (Pyridoxine HCl) 0.5mg / L, thiamine hydrochloride (Thiamine HCl) 0.1mg / 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] Thidiazuron (TDZ), CAS No.: 51707-55-2, structural formula:

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

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

[0071] Example 1 Establishment of sterile culture system

[0072] Select healthy semi-lignified stem segments ( Figure 1 Figure a), cut off the leaves, cut into 1-2 cm stem segments with buds according to the method of one node and one bud, soak and stir with detergent solution, gently brush the axillary buds of the stem segments with a soft brush, rinse with running water for 15 minutes to complete the initial disinfection of the explant. Then place it in an ultra-clean workbench, soak and disinfect it with 75% alcohol for 30 seconds, wash it with sterile water 3-5 times, soak and disinfect it with 1% sodium hypochlorite for 3 minutes, and wash it with sterile water 3-5 times. The disinfected explant was inoculated into the primary culture medium for 3 days of dark culture. After the end, it was transferred to the primary culture under the conditions of light intensity of 1500-2500lx, light cycle of 16h / d, and temperature of 23°C. After 30 days of culture, sterile seedlings were obtained ( Figure 1 (b).

[0073] Example 2 Induction and expansion culture of absorption surface

[0074] 1. Experimental Methods

[0075] The sterile sprouts obtained in Example 1 were cut into 1 cm sterile stem segments with one bud per node, and the sterile stem segments with buds were horizontally inoculated on absorption surface induction medium with different concentrations of TDZ (0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg / L) for absorption surface induction culture ( Figure 1 (Figure c) was used to compare the effects of different concentrations of TDZ on the absorptive surface induction of stem segments with buds and to determine the most suitable absorptive surface induction culture medium.

[0076] Subsequently, the stem segments with well-formed enlarged absorption surface were inoculated into absorption surface enlargement medium with different concentrations of 6-BA (1, 3, 5, 7, 9 mg / L) for absorption surface enlargement culture. The effects of different concentrations of 6-BA on the absorption surface enlargement were compared to determine the most suitable absorption surface enlargement medium.

[0077] The culture conditions for absorption surface induction culture and absorption surface expansion culture are as follows: the culture temperature is 23±2°C, the light conditions are 1500-2500lx, and the light duration is 16 hours per day.

[0078] Absorption surface induction medium components: MS medium + TDZ (0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg / L) + 3% sucrose + 0.7% agar; pH value is 5.8-6.0.

[0079] Absorption surface expansion culture medium components: MS culture medium + 6-BA (1, 3, 5, 7, 9 mg / L) + 3% sucrose + 0.7% agar; pH value is 5.8-6.0.

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

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

[0082] 2. Experimental Results

[0083] The results of the effects of different concentrations of TDZ on the induction of the absorption surface of stem segments with buds are shown in Table 1. As can be seen from Table 1, with the increase of TDZ concentration, the induction rate of the absorption surface showed a trend of first increasing and then decreasing. When the TDZ concentration was 1.5 mg / L, the induction effect on the absorption surface of axillary buds was the best. Both low and high concentrations of TDZ could cause axillary buds to germinate and significantly reduce the induction rate of the absorption surface ( Figure 1 During the experiment, it was found that after the primary culture, the stem segments with buds were inoculated into the absorption surface induction medium and the absorption surface was formed after about 21 days of culture.

[0084] The results of the effects of different concentrations of 6-BA on the expansion culture of the absorption surface are shown in Table 2. As can be seen from Table 2, with the increase of 6-BA concentration, the expansion rate of the absorption surface first increases and then decreases. 6-BA concentrations of 3-7 mg / L can expand the absorption surface. When the concentration of 6-BA is 5 mg / L, the expansion efficiency of the absorption surface is the highest, which is 77.79%, and the growth state is the best, which is a green expansion ball. Therefore, the appropriate concentration of 6-BA for the expansion development culture of the absorption surface is 5 mg / L ( Figure 1 Figure e). The culture time of the absorbing surface swelling culture medium is 20-30 days.

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

[0086]

[0087] Table 2 Effects of different concentrations of 6-BA on absorption surface expansion culture

[0088]

[0089] Example 3 Adventitious bud induction and elongation culture

[0090] 1. Experimental Methods

[0091] The stem segments with enlarged absorption surfaces after culture in Example 2 were transferred to adventitious bud induction 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 adventitious bud induction culture (culture time was 15-25 days) to induce the formation of adventitious bud clusters, and the effects of different hormone combinations on adventitious bud induction were compared to determine the most suitable adventitious bud induction medium.

[0092] Subsequently, the adventitious bud clusters were transferred to 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 adventitious bud elongation culture (culture time was 20-30 days), and the effects of different hormone combinations on adventitious bud elongation were compared to determine the optimal adventitious bud elongation medium.

[0093] Components of adventitious bud induction medium: 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: 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] 2. Experimental Results

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

[0097] As shown in Table 4, when the hormone combination is 0.5mg / L 6-BA and 0.05mg / L NAA, the adventitious bud elongation culture effect is the best, and the plant grows vigorously, with an average seedling height of 4.03cm. At the same time, it can 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 ratios ( Figure 1 h figure).

[0098] Table 3 Effects of different ratios of plant hormone combinations on adventitious bud induction

[0099]

[0100] Table 4 Effects of different ratios of plant hormone combinations on adventitious bud elongation

[0101]

[0102]

[0103] Example 4 Rooting Culture

[0104] 1. Experimental Methods

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

[0106] Six rooting induction media with different formulations were set up as different treatments;

[0107] The root induction medium formula for treatment 1 was: 1 / 2MS medium + 0.1 mg / L NAA + 0.7 mg / LAC + 3% sucrose + 0.7% agar;

[0108] The formula of the root induction medium for treatment 2 was: 1 / 2MS medium + 0.2 mg / L NAA + 0.7 mg / LAC + 3% sucrose + 0.7% agar;

[0109] The root induction medium formula for treatment 3 was: 1 / 2MS medium + 0.3 mg / L NAA + 0.7 mg / LAC + 3% sucrose + 0.7% agar;

[0110] The root induction medium formula for treatment 4 was: MS medium + 0.1 mg / L NAA + 0.7 mg / L AC + 3% sucrose + 0.7% agar;

[0111] The root induction medium formula for treatment 5 was: MS medium + 0.2 mg / L NAA + 0.7 mg / L AC + 3% sucrose + 0.7% agar;

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

[0113] 2. Experimental Results

[0114] As shown in Table 5, the induction rate of adventitious roots in 1 / 2MS medium is generally higher than that in MS medium, so 1 / 2MS medium is more conducive to the induction of adventitious roots in seedlings. Different concentrations of NAA can induce adventitious roots. With the increase of NAA concentration, the rooting rate of seedlings first increases and then decreases. When the NAA concentration is 0.2 mg / L, the rooting rate of seedlings is the highest, which is 86.67%. The induced adventitious roots are relatively strong and have more lateral roots ( Figure 1 (Figure 1).

[0115] Table 5 Effects of different treatments of root induction medium on root induction of seedlings

[0116]

[0117]

[0118] Example 5 Hardening and Transplanting

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

[0120] The Dioscorea zingiberensis tissue culture seedlings obtained by root induction in Example 4 were cultured in a culture room with the cover opened for 3-5 days. Before transplanting, the tissue culture seedlings were divided into individual plants with complete roots, stems and leaves. The individual seedlings were transferred to a transplant culture container with a transparent plastic cover and hardened in an incubator at a temperature of 23-25° C. and a humidity of 75-85%. The transplanted seedlings were able to grow and germinate normally, indicating that the transplant survived.

[0121] Comparative Example 1

[0122] The sterile sprouts obtained in Example 1 were cut into 1 cm sterile stem segments with buds according to one node and one bud, and the sterile stem segments with buds were inoculated into subculture medium for subculture (without absorption surface induction), and the subculture was subcultured every 30 days.

[0123] The results of subculture of sterile stem segments with buds are shown in Table 6. It can be seen from Table 6 that with the increase in the number of subcultures, the average number of buds and the average plant height both increased, and the average number of buds and the average plant height of the third subculture were the highest. In the first subculture, most of the buds became lighter in color, from the dark green in the primary culture process to light green, the growth of the buds became worse, and the seedlings were thin and weak. After the second subculture, the thin and weak buds stopped growing and gradually turned white, that is, the vitrification phenomenon of the buds occurred ( Figure 3 ), some sprouts with good growth will gradually show different degrees of browning ( Figure 4 ), the growth weakened. After three subcultures, the seedlings tended to be stable, but the proliferation coefficient was slow. During the subculture process, some seedlings grew poorly or even stopped growing because they could not adapt to the tissue culture environment, resulting in poor proliferation effect. Continuous subculture is required to make the growth of the seedlings tend to be stable. Experimental data show that at least 3 subcultures are required for the plants to grow normally.

[0124] Table 6 Effect of subculture times on subcultured sprouts

[0125]

[0126] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Application of MS medium containing thidiazuron and MS medium containing 6-benzylaminopurine in in vitro rapid propagation of Dioscorea zingiberensis.

2. The use according to claim 1, characterized in that: The concentration of thidiazuron in the MS culture medium containing thidiazuron is 1.0-1.5 mg / L, and the concentration of 6-benzylaminopurine in the MS culture medium containing 6-benzylaminopurine is 3-7 mg / L.

3. A method for in vitro rapid propagation of Dioscorea zingiberensis, characterized in that: Subculture is replaced by: inoculating the stem segments with buds of Dioscorea zingiberensis into an absorption surface induction medium for absorption surface induction, wherein the absorption surface induction medium comprises MS medium and thidiazuron.

4. The method according to claim 3, characterized in that: The concentration of thidiazuron in the absorption surface induction culture medium is 1.0-1.5 mg / L.

5. The method according to claim 3 or 4, characterized in that: The stem segments induced by the absorption surface 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.

6. The method according to claim 5, characterized in that: The concentration of 6-benzylaminopurine in the absorption surface expansion medium is 3-7 mg / L.

7. The method according to any one of claims 3 to 6, characterized in that: The steps include: (1) sterilizing the stem segments with buds of Dioscorea zingiberensis and conducting primary culture; (2) inoculating the stem segments after the primary culture in step (1) into an absorption surface induction medium for absorption surface induction; (3) inoculating the stem segments induced by the absorption surface in step (2) into the absorption surface expansion culture medium for absorption surface expansion culture; (4) taking the expanded absorption surface formed in step (3) to carry out adventitious bud induction culture and elongation culture to obtain adventitious bud clusters; (5) taking the adventitious bud clusters of step (4) for rooting culture to obtain Dioscorea zingiberensis tissue culture seedlings.

8. The method according to claim 7, characterized in that: The culture medium components used in the adventitious bud induction culture include MS culture medium, 6-benzylaminopurine, naphthaleneacetic acid, sucrose and agar; the culture medium components used in the adventitious bud elongation culture include MS culture medium, 6-benzylaminopurine, naphthaleneacetic acid, activated carbon, sucrose and agar.

9. The method according to claim 7, characterized in that: The culture medium components used in the rooting culture include 1 / 2MS culture medium, naphthylacetic acid, activated carbon, sucrose and agar.

10. The use according to claim 1 or 2 or the method according to any one of claims 3 to 9, characterized in that: The MS culture 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 ethylenediaminetetraacetic acid, niacin, vitamin B6, vitamin B1, inositol and glycine.

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