Method for inducing adventitious roots of paris polyphylla var. Yunnanensis

Abstract

The invention relates to the technical field of paris polyphylla var. Yunnanensis tissue culture, in particular to a paris polyphylla var. Yunnanensis adventitious root induction method which comprises the following steps: S1, establishing a paris polyphylla var. Yunnanensis sterile system; s2, preparing sterile seedlings of paris polyphylla var. Yunnanensis; s3, inducing the callus of the paris polyphylla var. Yunnanensis; s4, proliferation and differentiation of the adventitious roots of the paris polyphylla. According to the method, the problems that the existing paris polyphylla is long in growth cycle, cannot meet market requirements, and is limited in callus induction proliferation, so that adventitious roots of the paris polyphylla are difficult to induce are solved.

Description

Technical Field

[0001] The invention relates to the technical field of Paris polyphylla tissue culture, and more particularly to a method for inducing adventitious roots of Paris polyphylla. Background Art

[0002] Paris polyphylla var. Yunnanensis, also known as Paris polyphylla var. Yunnanensis, belongs to the Veratrum family (Melanthiaceae) of the Liliales order. It is also known as Caoheche, Dujiaolian, and Qiyeyizhihua. The rhizome of Paris polyphylla can be used as medicine and is a precious Chinese medicinal material. It was recorded as early as in the "Shennong Bencao Jing" and named "Zhaoxiu". It has the effects of clearing away heat and detoxification, reducing swelling and relieving pain, stopping bleeding and relieving pain. It can treat symptoms such as bruises, snake bites, convulsions, etc. It is an important raw material for Chinese patent medicines such as Yunnan Baiyao, Ji Desheng Snake Tablets, Gongxuening and Reduqing.

[0003] Dendrobium officinale is shade-tolerant and afraid of frost and direct sunlight. It is suitable for growing in evergreen broad-leaved forests, bamboo forests, shrubs or shaded areas of grass slopes and moist valleys at an altitude of 800-3100m.

[0004] Paris polyphylla takes a long time to grow, and under natural conditions, it needs to grow for more than ten years before it can be harvested. Due to the excessive excavation and development of wild Paris polyphylla in recent years, it is now extremely endangered. Paris polyphylla is widely used and is an important raw material in the fields of medicine, health care and cosmetics. With the increasing demand for Paris polyphylla, it is obviously difficult to meet the market demand for Paris polyphylla through wild collection and artificial cultivation.

[0005] Adventitious roots refer to the roots that grow on plant organs when plants are flooded or mechanically damaged, including callus differentiation and direct production. Adventitious roots can expand the area of ​​nutrient absorption, grow rapidly in a short period of time, and form a large number of roots that are easier to collect and have better stability. In addition, adventitious root cultivation is not restricted by geography and climate, occupies less space, is convenient for large-scale factory production, and saves land resources. The adventitious root products obtained by cultivation are controllable, high-quality, free of heavy metal pesticide residues, and meet national food standards.

[0006] In the field of plant organ culture, the research on adventitious roots of medicinal plants has always been a key topic, and a series of achievements have been made, and more and more large-scale industrialization has been achieved. Ginseng, American ginseng, sea cucumber, Panax notoginseng, etc. have always been the hot spots of adventitious root research, and have been used to produce secondary metabolites and put into large-scale production. In addition to the research on adventitious roots of the above-mentioned major medicinal materials, there are also explorations and discussions on other medicinal plants. The adventitious root culture systems of Polygonum multiflorum, Echinacea, Cornus officinalis, Tripterygium wilfordii, Hypericum perforatum, Bupleurum chinense, Clematis chinensis, etc. have been established.

[0007] Inducing adventitious roots of Paris polyphylla through tissue culture technology can not only achieve rapid reproduction of Paris polyphylla, but also effectively protect and utilize this precious medicinal plant resource. Due to the slow growth of Paris polyphylla and the limited proliferation of callus tissue, it is extremely difficult to induce adventitious roots of Paris polyphylla and there are no successful cases. Therefore, it is urgent to develop a method for inducing adventitious roots of Paris polyphylla to solve the above technical problems. Summary of the invention

[0008] The invention aims to solve the problems of long growth period and large market demand of Paris polyphylla, and provides a method for inducing adventitious roots of Paris polyphylla.

[0009] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0010] In a first aspect of the present invention, a method for inducing adventitious roots of Paris polyphylla is provided, comprising the following steps:

[0011] S1. Establishment of a sterile system of Paris polyphylla: Take Paris polyphylla stem nodes with buds and inoculate them in a sterile system culture medium to obtain Paris polyphylla explants; the Paris polyphylla sterile system culture medium is a modified MS solid culture medium with 0.02-0.1 mg / L antibiotics added;

[0012] S2. Preparation of sterile seedlings of Paris polyphylla: The Paris polyphylla explants obtained in S1 were transferred to Paris polyphylla sterile seedling culture medium to obtain Paris polyphylla sterile seedlings; the Paris polyphylla sterile seedling culture medium was a modified MS solid medium in which 1-3 mg / L 6-BA, 0.1-1.0 mg / L IAA, 0.1-0.5 mg / L KT and 0.05-0.2 mg / L ZT were added;

[0013] S3. Induction of Paris polyphylla callus: Cut Paris polyphylla fibrous roots of sterile Paris polyphylla seedlings obtained in S2 and inoculate them in Paris polyphylla callus induction medium to obtain Paris polyphylla callus; the Paris polyphylla callus induction medium is a modified MS solid medium with 0.5-2 mg / L 2,4-D, 0.2-1.0 mg / L 6-BA, 0.05-0.2 mg / L KT and 0.05-0.2 mg / L BR added;

[0014] S4. Proliferation and differentiation of adventitious roots of Paris polyphylla: The Paris polyphylla callus obtained in S3 was transferred to Paris polyphylla adventitious root proliferation and differentiation medium to obtain a large number of Paris polyphylla adventitious roots; the Paris polyphylla adventitious root proliferation and differentiation medium is a modified MS solid medium with 1-2 mg / L 2,4-D, 3-5 mg / L IBA, 0.2-1.0 mg / L NAA, 0.1-0.2 mg / L KT, 0.05-0.2 mg / L BR added;

[0015] The improved MS solid culture medium is the MS solid culture medium to which trace elements and macroelements are added.

[0016] In a preferred embodiment, the antibiotic is one or more of penicillins, aminoglycosides, quinolones, macrolides, cephalosporins and tetracyclines.

[0017] In a preferred embodiment, the improved MS solid medium is to add NH3 + and NO3 - The mass molar ratio of PO4 was adjusted to 1:(4-6), the total nitrogen source concentration was adjusted to 60-70mmol / L, and the 3- The concentration was adjusted to 20-25mmol / L, Mn 2+ The concentration was adjusted to 0.1-0.5mmol / L, Zn + The concentration was adjusted to 0.01-0.2mmol / L, BO3 3- The concentration was adjusted to 0.01-0.1mmol / L, Co 2+ The concentration is adjusted to 0.01-0.1 mmol / L.

[0018] In a preferred embodiment, steps S1, S3 and S4 are dark cultured at a temperature of 22-24°C, the culture time of step S1 is 2 weeks, the culture time of step S3 is 1 month, and the culture time of step S4 is 1.5 months.

[0019] In the preferred embodiment, step S2 is light culture, and the conditions of the light culture are light intensity 1200-1600 lx, light time 12h / d, temperature 24-26°C during light, temperature 22-24°C in darkness, and culture time 1 month.

[0020] In a preferred embodiment, the Paris polyphylla stem nodes with buds in step S1 are disinfected and then inoculated into a sterile culture medium.

[0021] In a preferred embodiment, the disinfection treatment is carried out in one or more solutions of alcohol, mercuric chloride and sodium hypochlorite in sequence or crosswise, the alcohol disinfection time is 50 seconds, the mercuric chloride disinfection time is 10 to 20 minutes, and the sodium hypochlorite disinfection time is 10 to 20 minutes.

[0022] In a second aspect of the present invention, a combined culture medium for inducing adventitious roots of Paris polyphylla is provided, comprising the following four culture media:

[0023] (1) a sterile system culture medium for culturing Paris polyphylla explants; the Paris polyphylla sterile system culture medium is a modified MS solid culture medium with 0.02-0.1 mg / L of antibiotics added;

[0024] (2) a culture medium for preparing sterile seedlings of Paris polyphylla; the culture medium for sterile seedlings of Paris polyphylla is a modified MS solid culture medium with 1-3 mg / L 6-BA, 0.1-1.0 mg / L IAA, 0.1-0.5 mg / L KT, and 0.05-0.2 mg / L ZT added thereto;

[0025] (3) a Paris polyphylla callus induction medium for inducing Paris polyphylla callus; the Paris polyphylla callus induction medium is a modified MS solid medium with 0.5-2 mg / L 2,4-D, 0.2-1.0 mg / L 6-BA, 0.05-0.2 mg / L KT and 0.05-0.2 mg / L BR added thereto;

[0026] (4) a Paris polyphylla adventitious root proliferation and differentiation medium for proliferation and differentiation of Paris polyphylla adventitious roots; the Paris polyphylla adventitious root proliferation and differentiation medium is a modified MS solid medium with 1-2 mg / L 2,4-D, 3-5 mg / L IBA, 0.2-1.0 mg / L NAA, 0.1-0.2 mg / L KT, and 0.05-0.2 mg / L BR added thereto;

[0027] The improved MS solid culture medium is the MS solid culture medium to which trace elements and macroelements are added.

[0028] In a preferred embodiment, the improved MS solid medium is to add NH3 + and NO3 - The mass molar ratio of PO4 was adjusted to 1:(4-6), the total nitrogen source concentration was adjusted to 60-70mmol / L, and the 3- The concentration was adjusted to 20-25mmol / L, Mn 2+ The concentration was adjusted to 0.1-0.5mmol / L, Zn + The concentration was adjusted to 0.01-0.2mmol / L, BO3 3- The concentration was adjusted to 0.01-0.1mmol / L, Co 2+ The concentration was adjusted to 0.01-0.1mmol / L.

[0029] In a preferred embodiment, the antibiotic is one or more of penicillins, aminoglycosides, quinolones, macrolides, cephalosporins and tetracyclines.

[0030] Beneficial effects of the present invention

[0031] 1. The present invention provides a modified MS solid culture medium suitable for growing Paris polyphylla tissue culture seedlings. The survival rate of Paris polyphylla tissue culture seedlings using MS solid culture medium is 0, while the survival rate using modified MS solid culture medium reaches 65.8%.

[0032] 2. The present invention uses an improved culture medium for sterile seedlings of Paris polyphylla to obtain sterile seedlings with high vitality. Before the addition of plant hormones, Paris polyphylla sterile seedlings survived for several months and maintained their status quo. Through experimental observations, it was found that there was dormancy inhibition. Through the exploration of the types and proportions of plant hormones and growth regulators, an improved culture medium for sterile seedlings of Paris polyphylla was invented to successfully break the dormancy inhibition and grow into complete plants.

[0033] 3. The present invention uses an improved callus induction medium of Paris polyphylla to induce callus using fibrous roots as explants. Different organs of Paris polyphylla sterile seedlings were selected for callus induction experiments, such as leaves, stem segments, fibrous roots and tubers, but the use of improved MS solid medium failed to induce successfully. By selecting the appropriate types and proportions of plant hormones and growth regulators, an improved callus induction medium of Paris polyphylla was invented, and the fibrous roots were used as explants for successful induction, with an induction rate of up to 63.2%.

[0034] 4. The present invention uses an improved adventitious root proliferation and differentiation medium to obtain adventitious roots of Paris polyphylla. Paris polyphylla callus is inoculated on the improved adventitious root proliferation and differentiation medium, and Paris polyphylla adventitious roots can be successfully proliferated and differentiated, with a proliferation coefficient of 2-3 times. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The sterile seedlings of Paris polyphylla obtained in Example 2;

[0036] Figure 2 The Paris polyphylla callus obtained in Example 2;

[0037] Figure 3 It is the adventitious root of Paris polyphylla obtained in Example 2. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0039] Detailed Overview of the Invention

[0040] The stem segments with buds of Paris polyphylla used in the experimental process of the present invention were purchased from Yunnan and were identified as Paris polyphylla after species identification. Other raw materials can be obtained through commercial channels unless otherwise specified. The experimental steps of the present invention are all carried out using conventional experimental operations and experimental conditions unless otherwise specified.

[0041] A method for inducing adventitious roots of Paris polyphylla, comprising the following steps:

[0042] S1. Establishment of the sterile system of Paris polyphylla;

[0043] S2. Preparation of sterile seedlings of Paris polyphylla;

[0044] S3. Induction of callus tissue of Paris polyphylla;

[0045] S4. Proliferation and differentiation of adventitious roots of Paris polyphylla.

[0046] Specifically, a method for inducing adventitious roots of Paris polyphylla comprises the following steps:

[0047] S1. Establishment of a sterile system for Paris polyphylla: Cut the stem nodes with buds from Paris polyphylla, sterilize them and inoculate them in a sterile culture medium and culture them in the dark for 14 days to obtain Paris polyphylla explants.

[0048] S2. Preparation of sterile seedlings of Paris polyphylla: The Paris polyphylla explants obtained in S1 were transferred to a Paris polyphylla sterile seedling culture medium and cultured in light for 30 days to obtain Paris polyphylla sterile seedlings.

[0049] S3. Induction of Paris polyphylla callus: Cut the fibrous roots of the Paris polyphylla sterile seedlings obtained in S2 and inoculate them into Paris polyphylla callus induction medium and culture them in the dark for 30 days to obtain Paris polyphylla callus.

[0050] S4. Proliferation and differentiation of adventitious roots of Paris polyphylla: The Paris polyphylla callus induced by S3 was transferred to Paris polyphylla adventitious root proliferation and differentiation medium and cultured in the dark for 45 days to obtain a large number of Paris polyphylla adventitious roots.

[0051] The dark culture temperature of steps S1, S3 and S4 is preferably 22-24°C.

[0052] The conditions for light culture in step S2 are preferably light intensity of 1200-1600 lx, light time of 12 h / d, temperature of 24-26° C. during light, and temperature of 22-24° C. during darkness.

[0053] The above-mentioned sterile system for Paris polyphylla was established by using alcohol, mercuric chloride and sodium hypochlorite for disinfection in sequence or crosswise.

[0054] The above-mentioned sterile system culture medium of Paris polyphylla is a modified MS solid culture medium with a certain proportion of antibiotics added, the added amount is 0.02-0.1 mg / L, and the maximum amount does not exceed 0.1 mg / L. The added antibiotics include one or more of penicillin antibiotics, aminoglycoside antibiotics, quinolone antibiotics, macrolide antibiotics, cephalosporin antibiotics and tetracycline antibiotics. The experimental results showed that the above antibiotics could inhibit the endophytes of Paris polyphylla and obtain sterile Paris polyphylla seedlings. The contamination rates were 48.6% for the blank control group, 10.6% for penicillins, 12.2% for aminoglycosides, 11.9% for quinolones, 12.7% for macrolides, 11.5% for cephalosporins and 12.1% for tetracyclines. Penicillins and cephalosporins had little effect on the growth of Paris polyphylla. The survival rates on the modified MS solid culture medium were 18.3% for the blank control group, 48.2% for penicillins, 25.9% for aminoglycosides, 24.3% for quinolones, 19.6% for macrolides, 51.1% for cephalosporins and 21.7% for tetracyclines.

[0055] The above-mentioned Paris polyphylla sterile seedling culture medium is a modified MS solid culture medium with 1-3 mg / L 6-BA, 0.1-1.0 mg / L IAA, 0.1-0.5 mg / L KT and 0.05-0.2 mg / L ZT added thereto.

[0056] The above-mentioned Paris polyphylla callus induction medium is a modified MS solid medium with 0.5-2 mg / L 2,4-D, 0.2-1.0 mg / L 6-BA, 0.05-0.2 mg / L KT and 0.05-0.2 mg / L BR added thereto.

[0057] The above-mentioned Paris polyphylla adventitious root proliferation and differentiation medium is a modified MS solid medium with 1-2 mg / L 2,4-D, 3-5 mg / L IBA, 0.2-1.0 mg / L NAA, 0.1-0.2 mg / L KT, and 0.05-0.2 mg / L BR added thereto.

[0058] The above-mentioned improved MS solid medium is a medium in which certain macroelements and trace elements are added exogenously on the basis of the basic medium. The improved MS solid medium is a medium in which NH3 + and NO3 - The mass molar ratio was adjusted to 1:(4-6), the total nitrogen source concentration was adjusted to 60-70mmol / L, PO4 3- The concentration was adjusted to 20-25mmol / L, Mn 2+ The concentration was adjusted to 0.1-0.5mmol / L, Zn +The concentration was adjusted to 0.01-0.2mmol / L, BO3 3- The concentration was adjusted to 0.01-0.1mmol / L, Co 2+ The concentration was adjusted to 0.01-0.1mmol / L.

[0059] Example 1 Study on the effect of antibiotics on Paris polyphylla explants

[0060] The culture of Paris polyphylla explants comprises the following steps: cutting multiple Paris polyphylla stem sections with buds, disinfecting them with 75% alcohol (50s) and mercuric chloride (20min) in sequence, and then inoculating them in modified MS solid culture medium with different types of antibiotics added, and culturing them in the dark for 14 days at a temperature of 22-24° C. The types of antibiotics added, the amount of antibiotics added, and the culture results of Paris polyphylla explants are shown in Tables 1-3.

[0061] The above-mentioned improved MS solid medium: on the basis of MS solid medium, NH3 + and NO3 - The mass molar ratio was adjusted to 1:4, the total nitrogen source concentration was adjusted to 60mmol / L, PO4 3- The concentration was adjusted to 20mmol / L, Mn 2+ The concentration was adjusted to 0.1mmol / L, Zn + The concentration was adjusted to 0.01mmol / L, BO3 3- The concentration was adjusted to 0.01mmol / L, Co 2+ The concentration was adjusted to 0.01 mmol / L.

[0062] Table 1 Effects of adding 0.02 mg / L antibiotics on the contamination rate and survival rate of Paris polyphylla explants

[0063] Types of antibiotics Antibiotics added Contamination rate Survival rate Blank control group — 48.6％ 18.3％ Penicillins 0.02mg / L 12.1％ 46.2％ Aminoglycosides 0.02mg / L 13.2％ 23.7％ Quinolones 0.02mg / L 12.4％ 21.4％ Macrolides 0.02mg / L 12.7％ 16.3％ Cephalosporins 0.02mg / L 11.5％ 48.7％ Tetracyclines 0.02mg / L 12.3％ 19.7％

[0064] Table 2 Effects of adding 0.05 mg / L antibiotics on the contamination rate and survival rate of Paris polyphylla explants

[0065]

[0066]

[0067] Table 3 Effects of adding 0.1 mg / L antibiotics on the contamination rate and survival rate of Paris polyphylla explants

[0068] Types of antibiotics Antibiotics added Contamination rate Survival rate Blank control group — 48.6％ 18.3％ Penicillins 0.1mg / L 10.6％ 48.2％ Aminoglycosides 0.1mg / L 12.2％ 25.9％ Quinolones 0.1mg / L 11.9％ 24.3％ Macrolides 0.1mg / L 12.9％ 19.6％ Cephalosporins 0.1mg / L 12.5％ 51.1％ Tetracyclines 0.1mg / L 12.1％ 21.7％

[0069] As shown in Table 1-3, the contamination rate of the Paris polyphylla tissue culture seedlings obtained by culturing in the modified MS medium without adding antibiotics was as high as 48.6%, and the contamination rate of the Paris polyphylla tissue culture seedlings obtained by culturing in the modified MS medium with 0.02-0.1 mg / L antibiotics was below 14%. The experimental results show that antibiotics can effectively inhibit the endophytes of Paris polyphylla, thereby obtaining sterile Paris polyphylla explants. Among them, penicillin antibiotics and cephalosporin antibiotics have little effect on the growth of Paris polyphylla explants, and the survival rates of Paris polyphylla explants are above 46.2% and 48.7%, respectively.

[0070] Example 2 Inducing adventitious roots of Paris polyphylla

[0071] A method for inducing adventitious roots of Paris polyphylla, comprising the following steps:

[0072] S1. Establishment of the sterile system of Paris polyphylla: Cut the stem nodes with buds of Paris polyphylla, disinfect them with 75% alcohol (50s) and mercuric chloride (20min) respectively, and then inoculate them in the sterile system culture medium for dark culture for 14 days at 22°C. The sterile seedling system culture medium is a modified MS solid culture medium with 0.02mg / L penicillin and 0.1mg / L cephalexin added. The contamination rate of Paris polyphylla explants obtained after 14 days was 9.82%, and the survival rate was 62.7%.

[0073] S2. Preparation of sterile seedlings of Paris polyphylla: The Paris polyphylla explants obtained in S1 were transferred to Paris polyphylla sterile seedling culture medium for light culture for 30 days. The Paris polyphylla sterile seedling culture medium is a modified MS solid culture medium with 1mg / L 6-BA, 0.1mg / L IAA, 0.1mg / L KT and 0.05mg / L ZT added. The conditions for light culture are light intensity of 1200lx, light time of 12h / d, temperature of 24°C during light, and temperature of 22°C in the dark. After 30 days, complete Paris polyphylla sterile seedling plants were grown to obtain highly vigorous Paris polyphylla sterile seedlings.

[0074] S3. Induction of Paris polyphylla callus: The fibrous roots of Paris polyphylla sterile seedlings obtained in S2 were inoculated into Paris polyphylla callus induction medium and cultured in the dark for 30 days at 22°C. Paris polyphylla callus induction medium was a modified MS solid medium with 0.5 mg / L 2,4-D, 0.2 mg / L 6-BA, 0.05 mg / L KT and 0.05 mg / L BR added. After 30 days, the induction rate of Paris polyphylla callus was 61.8%.

[0075] S4. Proliferation and differentiation of adventitious roots of Paris polyphylla: The Paris polyphylla callus induced by S3 was transferred to Paris polyphylla adventitious root proliferation and differentiation medium and cultured in the dark for 45 days at 22°C. Paris polyphylla adventitious root proliferation and differentiation medium is a modified MS solid medium with 1mg / L 2,4-D, 3mg / L IBA, 0.2mg / L NAA, 0.1mg / L KT and 0.05mg / L BR added. The root growth coefficient of Paris polyphylla after 45 days is 2.4.

[0076] The above-mentioned improved MS solid medium: on the basis of MS solid medium, NH3 + and NO3 - The mass molar ratio was adjusted to 1:4, the total nitrogen source concentration was adjusted to 60mmol / L, PO4 3- The concentration was adjusted to 20mmol / L, Mn 2+ The concentration was adjusted to 0.1mmol / L, Zn + The concentration was adjusted to 0.01mmol / L, BO3 3- The concentration was adjusted to 0.01mmol / L, Co 2+ The concentration was adjusted to 0.01 mmol / L.

[0077] like Figures 1 to 3 They are respectively the sterile seedlings, callus tissue and adventitious roots of Paris polyphylla cultured by the method of the present invention, wherein the callus tissue and adventitious roots have not been studied, reported or shown in actual figures before. Figure 1 The high-vigor sterile seedlings obtained by culturing the Paris polyphylla sterile seedling culture medium in Example 2 have spreading green leaves and erect stems. Figure 2 The callus tissue selected for fibrous root induction in Example 2 is yellowish white and has a hard surface texture. Figure 3 These are adventitious roots differentiated from the callus in Example 2. The transparent root tips can be observed to be newly formed adventitious roots.

[0078] Example 3 Inducing adventitious roots of Paris polyphylla

[0079] A method for inducing adventitious roots of Paris polyphylla, comprising the following steps:

[0080] S1. Establishment of the sterile system of Paris polyphylla: Cut the stem nodes with buds from Paris polyphylla, disinfect them with 75% alcohol (50s) and sodium hypochlorite (20min) respectively, and then inoculate them in the sterile system culture medium for dark culture for 14 days at 23°C. The sterile seedling system culture medium is a modified MS solid culture medium with 0.05mg / L penicillin and 0.05mg / L cephalexin added. The contamination rate of Paris polyphylla explants obtained after 14 days was 8.4%, and the survival rate was 65.8%.

[0081] S2. Preparation of sterile seedlings of Paris polyphylla: The Paris polyphylla explants obtained in S1 were transferred to Paris polyphylla sterile seedling culture medium for light culture for 30 days. The Paris polyphylla sterile seedling culture medium is a modified MS solid culture medium with 2mg / L 6-BA, 0.5mg / L IAA, 0.2mg / L KT and 0.1mg / L ZT added. The conditions for light culture are light intensity of 1400lx, light time of 12h / d, temperature of 25℃ during light, and temperature of 23℃ in darkness. After 30 days, complete Paris polyphylla sterile seedling plants were grown to obtain highly vigorous Paris polyphylla sterile seedlings.

[0082] S3. Induction of Paris polyphylla callus: The fibrous roots of Paris polyphylla sterile seedlings obtained in S2 were inoculated into Paris polyphylla callus induction medium and cultured in the dark for 30 days at 23°C. Paris polyphylla callus induction medium was a modified MS solid medium with 1mg / L 2,4-D, 0.5mg / L 6-BA, 0.1mg / L KT, and 0.1mg / L BR added. After 30 days, the induction rate of Paris polyphylla callus was 63.2%.

[0083] S4. Proliferation and differentiation of adventitious roots of Paris polyphylla: The Paris polyphylla callus induced by S3 was transferred to Paris polyphylla adventitious root proliferation and differentiation medium and cultured in the dark for 45 days at 23°C. Paris polyphylla adventitious root proliferation and differentiation medium is a modified MS solid medium with 1mg / L 2,4-D, 4mg / L IBA, 0.5mg / L NAA, 0.1mg / L KT and 0.1mg / L BR added. The root growth coefficient of Paris polyphylla after 45 days is 3.1.

[0084] The above-mentioned improved MS solid medium: on the basis of MS solid medium, NH3 + and NO3 - The mass molar ratio was adjusted to 1:5, the total nitrogen source concentration was adjusted to 65mmol / L, PO4 3- The concentration was adjusted to 20mmol / L, Mn 2+ The concentration was adjusted to 0.3mmol / L, Zn + The concentration was adjusted to 0.1mmol / L, BO3 3- The concentration was adjusted to 0.05mmol / L, Co 2+ The concentration was adjusted to 0.05mmol / L.

[0085] Example 4 Inducing adventitious roots of Paris polyphylla

[0086] S1. Establishment of the sterile system of Paris polyphylla: Cut the stem nodes with buds from Paris polyphylla, disinfect them with 75% alcohol (50s), mercuric chloride (10min), and sodium hypochlorite (20min) in sequence, and then inoculate them in sterile system culture medium for dark culture for 14 days at 23°C. The sterile seedling system culture medium is a modified MS solid culture medium with 0.1mg / L penicillin and 0.02mg / L cephalexin added. The contamination rate of Paris polyphylla explants obtained after 14 days was 9.26%, and the survival rate was 63.4%.

[0087] S2. Preparation of sterile seedlings of Paris polyphylla: The Paris polyphylla explants obtained in S1 were transferred to Paris polyphylla sterile seedling culture medium for light culture for 30 days. The Paris polyphylla sterile seedling culture medium is a modified MS solid culture medium with 3mg / L 6-BA, 1.0mg / L IAA, 0.5mg / L KT and 0.2mg / L ZT added. The conditions for light culture are light intensity of 1600lx, light time of 12h / d, temperature of 26°C during light, and temperature of 24°C in the dark. After 30 days, complete Paris polyphylla sterile seedling plants were grown to obtain highly vigorous Paris polyphylla sterile seedlings.

[0088] S3. Induction of Paris polyphylla callus: The fibrous roots of Paris polyphylla sterile seedlings obtained in S2 were inoculated into Paris polyphylla callus induction medium and cultured in the dark for 30 days at 24°C. Paris polyphylla callus induction medium was a modified MS solid medium with 2 mg / L 2,4-D, 1.0 mg / L 6-BA, 0.2 mg / L KT and 0.2 mg / L BR added. After 30 days, the induction rate of Paris polyphylla callus was 62.7%.

[0089] S4. Proliferation and differentiation of adventitious roots of Paris polyphylla: The Paris polyphylla callus induced by S3 was transferred to Paris polyphylla adventitious root proliferation and differentiation medium and cultured in the dark for 45 days at 24°C. Paris polyphylla adventitious root proliferation and differentiation medium is a modified MS solid medium with 2mg / L 2,4-D, 5mg / L IBA, 0.5mg / L NAA, 0.2mg / L KT and 0.2mg / L BR added. The root growth coefficient of Paris polyphylla after 45 days was 2.8.

[0090] The above-mentioned improved MS solid medium: on the basis of MS solid medium, NH3 + and NO3 - The mass molar ratio was adjusted to 1:6, the total nitrogen source concentration was adjusted to 70mmol / L, PO4 3- The concentration was adjusted to 25mmol / L, Mn 2+ The concentration was adjusted to 0.5mmol / L, Zn + The concentration was adjusted to 0.2mmol / L, BO3 3- The concentration was adjusted to 0.1mmol / L, Co 2+The concentration was adjusted to 0.1 mmol / L.

[0091] Table 4 Effect of culture medium on the vitality of Paris polyphylla sterile seedlings

[0092] Implementation Cases Seedling time Seedling rate Seedling emergence Blank control (MS) — — No significant changes Example 1 26d 64.3％ Grows a single upright stem Example 2 24d 69.6％ Erect stems grow, and a few have clustered upright stems Example 3 27d 65.7％ Growing upright stems, rarely clustered upright stems

[0093] As shown in Table 4, the culture process of the blank control is to replace the sterile seedling culture medium of Paris polyphylla with MS solid culture medium, and the other processes and conditions are the same as steps S1-S2 of Example 2. It is observed that there is no obvious change in Paris polyphylla explants before and after cultivation, and MS solid culture medium cannot induce Paris polyphylla explants to grow and sprout. The time taken for Paris polyphylla explants of Examples 1-3 to sprout in the sterile seedling culture medium of Paris polyphylla was recorded respectively, and the emergence rate was calculated. The experimental results show that high-vitality sterile seedlings can be obtained by culturing the Paris polyphylla sterile seedling culture medium provided by the present invention, and the emergence rate of Examples 1-3 can reach more than 64%.

[0094] Comparative Example 1

[0095] The culture of Paris polyphylla explants comprises the following steps: cutting Paris polyphylla stem nodes with buds, disinfecting them with 75% alcohol (50s) and mercuric chloride (20min) in sequence, and then inoculating them in MS solid culture medium for dark culture for 14 days at a temperature of 22-24° C. The survival rate of Paris polyphylla explants obtained after 14 days is 0.

[0096] Comparative Example 2

[0097] The cultivation of sterile seedlings of Paris polyphylla includes the following steps:

[0098] S1. Establishment of the sterile system of Paris polyphylla: Cut several sections of Paris polyphylla with buds, sterilize them with 75% alcohol (50s) and mercuric chloride (20min) respectively, and then inoculate them in sterile system culture medium for 14 days. The sterile seedling system culture medium is a modified MS solid culture medium with 0.02mg / L penicillin and 0.1mg / L cephalexin added. The survival rate of Paris polyphylla explants obtained after 14 days is 62.7%.

[0099] S2. Preparation of sterile seedlings of Paris polyphylla: The Paris polyphylla explants obtained in S1 were transferred to the modified MS solid medium for light culture for 1 to 3 months. The conditions for light culture were light intensity of 1200 lx, light time of 12 h / d, temperature of 24-26 ° C during light, and temperature of 22-24 ° C during darkness. Experimental observations found that after several months, the growth of Paris polyphylla sterile seedlings was in a dormant inhibition state and could not grow further.

[0100] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for inducing adventitious roots of Paris polyphylla, characterized in that: The steps include: S1. Establishment of a sterile system of Paris polyphylla: Take Paris polyphylla stem nodes with buds and inoculate them in a sterile system culture medium to obtain Paris polyphylla explants; the Paris polyphylla sterile system culture medium is a modified MS solid culture medium with 0.02-0.1 mg / L antibiotics added; S2. Preparation of sterile seedlings of Paris polyphylla: The Paris polyphylla explants obtained in S1 were transferred to Paris polyphylla sterile seedling culture medium to obtain Paris polyphylla sterile seedlings; the Paris polyphylla sterile seedling culture medium was a modified MS solid medium in which 1-3 mg / L 6-BA, 0.1-1.0 mg / L IAA, 0.1-0.5 mg / L KT and 0.05-0.2 mg / L ZT were added; S3. Induction of Paris polyphylla callus: Cut Paris polyphylla fibrous roots of sterile Paris polyphylla seedlings obtained in S2 and inoculate them in Paris polyphylla callus induction medium to obtain Paris polyphylla callus; the Paris polyphylla callus induction medium is a modified MS solid medium with 0.5-2 mg / L 2,4-D, 0.2-1.0 mg / L 6-BA, 0.05-0.2 mg / L KT and 0.05-0.2 mg / L BR added; S4. Proliferation and differentiation of adventitious roots of Paris polyphylla: The Paris polyphylla callus obtained in S3 was transferred to Paris polyphylla adventitious root proliferation and differentiation medium to obtain a large number of Paris polyphylla adventitious roots; the Paris polyphylla adventitious root proliferation and differentiation medium is a modified MS solid medium with 1-2 mg / L 2,4-D, 3-5 mg / L IBA, 0.2-1.0 mg / L NAA, 0.1-0.2 mg / L KT, 0.05-0.2 mg / L BR added; The improved MS solid culture medium is the MS solid culture medium to which trace elements and macroelements are added.

2. The method for inducing adventitious roots of Paris polyphylla according to claim 1, characterized in that: The antibiotic is one or more of penicillins, aminoglycosides, quinolones, macrolides, cephalosporins and tetracyclines.

3. The method for inducing adventitious roots of Paris polyphylla according to claim 1, characterized in that: The improved MS solid medium is to add NH3 + and NO3 - The mass molar ratio of PO4 was adjusted to 1:(4-6), the total nitrogen source concentration was adjusted to 60-70mmol / L, and the 3- The concentration was adjusted to 20-25mmol / L, Mn 2+ The concentration was adjusted to 0.1-0.5mmol / L, Zn + The concentration was adjusted to 0.01-0.2mmol / L, BO3 3- The concentration was adjusted to 0.01-0.1mmol / L, Co 2+ The concentration is adjusted to 0.01-0.1 mmol / L.

4. The method for inducing adventitious roots of Paris polyphylla according to claim 1, characterized in that: Steps S1, S3 and S4 are dark cultures at a temperature of 22-24°C. The culture time of step S1 is 2 weeks, the culture time of step S3 is 1 month, and the culture time of step S4 is 1.5 months.

5. The method for inducing adventitious roots of Paris polyphylla according to claim 1, characterized in that: Step S2 is light culture, and the conditions of the light culture are light intensity 1200-1600lx, light time 12h / d, temperature 24-26°C during light, temperature 22-24°C in the dark, and culture time 1 month.

6. The method for inducing adventitious roots of Paris polyphylla according to claim 1, characterized in that: Step S1: the Paris polyphylla stem nodes with buds are disinfected and then inoculated into a sterile culture medium.

7. The method for inducing adventitious roots of Paris polyphylla according to claim 6, characterized in that: The disinfection treatment is to disinfect in one or more solutions of alcohol, mercuric chloride and sodium hypochlorite in sequence or crosswise, the alcohol disinfection time is 50 seconds, the mercuric chloride disinfection time is 10 to 20 minutes, and the sodium hypochlorite disinfection time is 10 to 20 minutes.

8. A combined culture medium for inducing adventitious roots of Paris polyphylla, characterized in that: The following four culture media are included: (1) a sterile system culture medium for culturing Paris polyphylla explants; the Paris polyphylla sterile system culture medium is a modified MS solid culture medium with 0.02-0.1 mg / L of antibiotics added; (2) a culture medium for preparing sterile seedlings of Paris polyphylla; the culture medium for sterile seedlings of Paris polyphylla is a modified MS solid culture medium with 1-3 mg / L 6-BA, 0.1-1.0 mg / L IAA, 0.1-0.5 mg / L KT, and 0.05-0.2 mg / L ZT added thereto; (3) a Paris polyphylla callus induction medium for inducing Paris polyphylla callus; the Paris polyphylla callus induction medium is a modified MS solid medium with 0.5-2 mg / L 2,4-D, 0.2-1.0 mg / L 6-BA, 0.05-0.2 mg / L KT and 0.05-0.2 mg / L BR added thereto; (4) a Paris polyphylla adventitious root proliferation and differentiation medium for proliferation and differentiation of Paris polyphylla adventitious roots; the Paris polyphylla adventitious root proliferation and differentiation medium is a modified MS solid medium with 1-2 mg / L 2,4-D, 3-5 mg / L IBA, 0.2-1.0 mg / L NAA, 0.1-0.2 mg / L KT, and 0.05-0.2 mg / L BR added thereto; The improved MS solid culture medium is the MS solid culture medium to which trace elements and macroelements are added.

9. The combined culture medium for inducing adventitious roots of Paris polyphylla according to claim 8, characterized in that: The improved MS solid medium is to add NH3 + and NO3 - The mass molar ratio of PO4 was adjusted to 1:(4-6), the total nitrogen source concentration was adjusted to 60-70mmol / L, and the 3- The concentration was adjusted to 20-25mmol / L, Mn 2+ The concentration was adjusted to 0.1-0.5mmol / L, Zn + The concentration was adjusted to 0.01-0.2mmol / L, BO3 3- The concentration was adjusted to 0.01-0.1mmol / L, Co 2+ The concentration was adjusted to 0.01-0.1mmol / L.

10. The combined culture medium for inducing adventitious roots of Paris polyphylla according to claim 8, characterized in that: The antibiotic is one or more of penicillins, aminoglycosides, quinolones, macrolides, cephalosporins and tetracyclines.

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