A method for inducing adventitious roots of paris polyphylla var. yunnanensis

Abstract

The present application relates to the tissue culture technical field of Yunnan rhizoma paridis, and particularly relates to a method for inducing adventitious roots of Yunnan rhizoma paridis, comprising the following steps: S1. establishing a sterile system of Yunnan rhizoma paridis; S2. preparing sterile seedlings of Yunnan rhizoma paridis; S3. inducing callus of Yunnan rhizoma paridis; S4. proliferating and differentiating adventitious roots of Yunnan rhizoma paridis. The present application overcomes the problems that the existing Yunnan rhizoma paridis has a long growth cycle, cannot meet market demand, callus induction and proliferation are limited, and thus it is difficult to induce adventitious roots of Yunnan rhizoma paridis.

Description

Technical Field

[0001] This invention relates to the field of Paris polyphylla tissue culture technology, and more specifically, to a method for inducing adventitious roots in Paris polyphylla. Background Technology

[0002] Paris polyphylla var. Yunnanensis, also known as Yunnan Paris polyphylla, belongs to the genus Paris of the family Melanthiaceae in the order Liliales. Other names include Caoheche, Dujiaolian, and Qiye Yizhihua. The rhizome of Paris polyphylla is used medicinally and is a valuable traditional Chinese medicine. It was recorded as early as the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) under the name "Zaoxiu." It has the effects of clearing heat and detoxifying, reducing swelling and relieving pain, and stopping bleeding and pain. It can treat symptoms such as traumatic pain, snake bites, and infantile convulsions. It is an important raw material for many traditional Chinese medicines, including Yunnan Baiyao, Jidesheng Snake Tablets, Gongxue Ning, and Reduqing.

[0003] Paris polyphylla is highly shade-tolerant but intolerant of frost and direct sunlight. It is suitable for growing in evergreen broad-leaved forests, bamboo forests, thickets, or shady grassy slopes and damp valleys at altitudes of 800-3100m.

[0004] Paris polyphylla has a long growth period, requiring more than ten years to mature under natural conditions before it can be harvested. Due to over-exploitation of wild Paris polyphylla in recent years, it is now critically endangered. Paris polyphylla has a wide range of applications and is an important raw material in the fields of medicine, health care, and cosmetics. With the increasing demand for Paris polyphylla, it is clearly difficult to meet the market demand 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 those differentiated from callus tissue and those directly produced. Adventitious roots can expand the nutrient absorption area, grow rapidly in a short period of time, form a large root system that is easier to collect, and have better stability. Moreover, adventitious root cultivation is not limited by region or climate, occupies less space, is convenient for large-scale industrial production, saves land resources, and the adventitious root products obtained are controllable, of high quality, free from heavy metal pesticide residues, and meet national food standards.

[0006] In the field of plant organ culture, the study of adventitious roots of medicinal plants has always been a key topic, yielding a series of results and increasingly achieving large-scale industrialization. Ginseng, American ginseng, Northeast sea cucumber, and Panax notoginseng have consistently been hot topics in adventitious root research, and are currently being used to produce secondary metabolites in large-scale production. Besides the above-mentioned major medicinal plants, research on adventitious roots of other medicinal plants is also underway. Adventitious root culture systems for Polygonum multiflorum, Chrysanthemum indicum, Cornus officinalis, Tripterygium wilfordii, St. John's wort, Bupleurum chinense, and Clematis chinensis have been established.

[0007] Inducing adventitious roots in Paris polyphylla through tissue culture not only enables rapid propagation of the plant but also effectively protects and utilizes this valuable medicinal plant resource. However, due to the slow growth of Paris polyphylla and the limited proliferation caused by callus induction, inducing adventitious roots has proven extremely difficult and there are no successful precedents. Therefore, there is an urgent need to develop a method for inducing adventitious roots in Paris polyphylla to solve these technical problems. Summary of the Invention

[0008] This invention aims to solve the problems of long growth cycle and high market demand for Paris polyphylla, and provides a method for inducing adventitious roots in Paris polyphylla.

[0009] To achieve the above objectives, the present invention is implemented through the following technical solution:

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

[0011] S1. Establishment of a sterile system for Paris polyphylla: Bud-bearing stem segments of Paris polyphylla were inoculated into a sterile culture medium to obtain Paris polyphylla explants; the sterile culture medium was a modified MS solid medium with 0.02-0.1 mg / L antibiotics added.

[0012] S2. Preparation of aseptic seedlings of Paris polyphylla: The explants of Paris polyphylla obtained in S1 were transferred to aseptic seedling culture medium to obtain aseptic seedlings of Paris polyphylla; the aseptic seedling culture medium was a modified MS solid medium supplemented 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;

[0013] S3. Induction of Paris polyphylla callus: The fibrous roots of Paris polyphylla obtained from the sterile seedlings in S2 were inoculated into Paris polyphylla callus induction medium to obtain Paris polyphylla callus; the Paris polyphylla callus induction medium was a modified MS solid medium supplemented 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.

[0014] S4. Proliferation and differentiation of adventitious roots of Paris polyphylla: The Paris polyphylla callus obtained in S3 was transferred to the Paris polyphylla adventitious root proliferation and differentiation medium for culture, resulting in a large number of Paris polyphylla adventitious roots; the Paris polyphylla adventitious root proliferation and differentiation medium was a modified MS solid medium supplemented 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;

[0015] The modified MS solid culture medium is MS solid culture medium with added trace elements and macro elements.

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

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

[0018] In the preferred embodiment, steps S1, S3 and S4 are carried out in the dark at a temperature of 22-24℃, with the culture time for step S1 being 2 weeks, the culture time for step S3 being 1 month, and the culture time for step S4 being 1.5 months.

[0019] In the preferred embodiment, step S2 is light culture, wherein the light culture conditions are: light intensity 1200-1600 lx, light duration 12 h / d, temperature 24-26℃ during light exposure, temperature 22-24℃ during darkness, and culture time 1 month.

[0020] In the preferred embodiment, in step S1, the stem segments of Paris polyphylla with buds are disinfected before being inoculated into a sterile culture medium.

[0021] In a preferred embodiment, the disinfection process involves sequentially or alternately disinfecting in one or more solutions of alcohol, mercuric chloride, and sodium hypochlorite, with the alcohol disinfection time being 50 seconds, the mercuric chloride disinfection time being 10-20 minutes, and the sodium hypochlorite disinfection time being 10-20 minutes.

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

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

[0024] (2) A sterile culture medium for preparing sterile Paris polyphylla seedlings; the sterile culture medium for Paris polyphylla seedlings is a modified MS solid medium supplemented 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;

[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 supplemented 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;

[0026] (4) A medium for the proliferation and differentiation of adventitious roots of Paris polyphylla; the medium for the proliferation and differentiation of adventitious roots of Paris polyphylla is a modified MS solid medium supplemented 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;

[0027] The modified MS solid culture medium is MS solid culture medium with added trace elements and macro elements.

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

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

[0030] Beneficial effects of the present invention

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

[0032] 2. This invention uses a modified aseptic seedling culture medium for Paris polyphylla to obtain highly vigorous aseptic seedlings. Before the addition of plant hormones, the aseptic seedlings of Paris polyphylla maintained their current state for several months while surviving. Experimental observation revealed dormancy inhibition. Through research on the types and proportions of plant hormones and growth regulators, a modified aseptic seedling culture medium for Paris polyphylla was invented, successfully breaking dormancy inhibition and allowing the seedlings to grow into complete plants.

[0033] 3. This invention uses a modified Paris polyphylla callus induction medium, employing fibrous roots as explants to induce callus. Callus induction experiments were conducted on different organs of aseptic Paris polyphylla seedlings, such as leaves, stem segments, fibrous roots, and tubers. Induction was unsuccessful using the modified MS solid medium. By selecting appropriate types and ratios of plant hormones and growth regulators, a modified Paris polyphylla callus induction medium was invented, successfully inducing callus using fibrous roots as explants, with an induction rate as high as 63.2%.

[0034] 4. This invention uses a modified adventitious root proliferation and differentiation medium to obtain adventitious roots of Paris polyphylla. By inoculating Paris polyphylla callus tissue onto the modified adventitious root proliferation and differentiation medium, adventitious roots of Paris polyphylla can be successfully obtained through proliferation and differentiation, with a proliferation coefficient of 2-3 times. Attached Figure Description

[0035] Figure 1 It is the aseptic seedling of Paris polyphylla obtained in Example 2;

[0036] Figure 2 This is the Paris polyphylla callus obtained in Example 2;

[0037] Figure 3 These are the adventitious roots of Paris polyphylla obtained in Example 2. Detailed Implementation

[0038] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0039] Detailed overview of the invention

[0040] The stem segments with buds used in the experiments of this invention were purchased locally in Yunnan and identified as Paris polyphylla. Other raw materials, unless otherwise specified, can be obtained through commercial channels. Unless otherwise specified, the experimental steps of this invention are carried out using conventional experimental operations and conditions.

[0041] A method for inducing adventitious roots in Paris polyphylla includes the following steps:

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

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

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

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

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

[0047] S1. Establishment of a sterile system for Paris polyphylla: Stem segments with buds were cut from Paris polyphylla, disinfected, and then inoculated into a sterile culture medium and cultured in the dark for 14 days to obtain Paris polyphylla explants.

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

[0049] S3. Induction of Paris polyphylla callus: The fibrous roots of the sterile Paris polyphylla seedlings obtained in S2 were inoculated into the Paris polyphylla callus induction medium and cultured in the dark for 30 days to obtain Paris polyphylla callus.

[0050] S4. Proliferation and differentiation of adventitious roots of Paris polyphylla: S3-induced Paris polyphylla callus 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 preferred dark culture temperature for steps S1, S3, and S4 is 22-24℃.

[0052] The preferred conditions for light culture in step S2 are: light intensity of 1200-1600 lx, light duration of 12 h / d, temperature of 24-26℃ during light exposure, and temperature of 22-24℃ during darkness.

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

[0054] The above-mentioned aseptic culture medium for *Paris polyphylla* is a modified MS solid medium with a certain proportion of antibiotics added. The amount added is 0.02-0.1 mg / L, and the maximum is no more than 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 all the above-mentioned antibiotics could inhibit the endophytic bacteria of Paris polyphylla to 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 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 aseptic seedling culture medium of Paris polyphylla is a modified MS solid medium supplemented 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.

[0056] The above-mentioned callus induction medium of Paris polyphylla is a modified MS solid medium supplemented 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.

[0057] The above-mentioned adventitious root proliferation and differentiation medium of Paris polyphylla is a modified MS solid medium supplemented 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.

[0058] The aforementioned modified MS solid medium is prepared by exogenously adding certain macro- and micro-elements to a basic culture medium. The modified MS solid medium is prepared by adding NH3 to the MS solid medium. + and NO3 - Adjust the molar ratio to 1:(4-6), adjust the total nitrogen source concentration to 60-70 mmol / L, and PO42-... 3- Adjust the concentration to 20-25 mmol / L, Mn 2+ Adjust the concentration to 0.1-0.5 mmol / L, Zn +Adjust the concentration to 0.01-0.2 mmol / L, BO3 3- Adjust the concentration to 0.01-0.1 mmol / L, Co 2+ Adjust the concentration to 0.01-0.1 mmol / L.

[0059] Example 1 Investigating the effects of antibiotics on Paris polyphylla explants

[0060] The culture of Paris polyphylla explants included the following steps: multiple stem segments with buds were cut and disinfected sequentially with 75% alcohol (50s) and mercuric chloride (20min), and then inoculated onto modified MS solid medium supplemented with different types of antibiotics and cultured in the dark for 14 days at 22-24℃. The types and amounts of antibiotics added and the culture results of the Paris polyphylla explants are shown in Tables 1-3.

[0061] The above-mentioned modified MS solid medium: Based on MS solid medium, NH3 is added + and NO3 - The mass molar ratio was adjusted to 1:4, the total nitrogen source concentration was adjusted to 60 mmol / L, and PO42- 3- The concentration was adjusted to 20 mmol / L, Mn 2+ Adjust the concentration to 0.1 mmol / L, Zn + Adjust the concentration to 0.01 mmol / L, BO3 3- Adjust the concentration to 0.01 mmol / L, Co 2+ Adjust the concentration to 0.01 mmol / L.

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

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

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

[0065]

[0066]

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

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

[0069] As shown in Tables 1-3, the contamination rate of *Paris polyphylla* tissue culture seedlings obtained from modified MS medium without antibiotics was as high as 48.6%, while the contamination rate of *Paris polyphylla* tissue culture seedlings obtained from modified MS medium with antibiotics added at 0.02-0.1 mg / L was all below 14%. The experimental results indicate that antibiotics can effectively inhibit endophytic bacteria in *Paris polyphylla*, thus obtaining sterile *Paris polyphylla* explants. Among them, penicillin and cephalosporin antibiotics had relatively little effect on the growth of *Paris polyphylla* explants, with survival rates of 46.2% and 48.7% or higher, respectively.

[0070] Example 2 Inducing adventitious roots of Paris polyphylla

[0071] A method for inducing adventitious roots in Paris polyphylla includes the following steps:

[0072] S1. Establishment of a sterile system for *Paris polyphylla*: Stem segments with buds were cut from *Paris polyphylla*, and sterilized sequentially with 75% alcohol (50 s) and mercuric chloride (20 min) before being inoculated into a sterile culture medium and cultured in the dark for 14 days at 22℃. The sterile seedling culture medium was a modified MS solid medium supplemented with 0.02 mg / L penicillin and 0.1 mg / L cephalexin. After 14 days, the contamination rate of the *Paris polyphylla* explants was 9.82%, and the survival rate was 62.7%.

[0073] S2. Preparation of aseptic seedlings of *Paris polyphylla*: The *Paris polyphylla* explants obtained in S1 were transferred to a *Paris polyphylla* aseptic seedling culture medium and cultured under light for 30 days. The *Paris polyphylla* aseptic seedling culture medium was a modified MS solid medium supplemented with 1 mg / L 6-BA, 0.1 mg / L IAA, 0.1 mg / L KT, and 0.05 mg / L ZT. The light culture conditions were: light intensity 1200 lx, light duration 12 h / d, temperature 24℃ during light exposure, and temperature 22℃ during darkness. After 30 days, complete *Paris polyphylla* aseptic seedlings were formed, yielding highly vigorous *Paris polyphylla* aseptic seedlings.

[0074] S3. Induction of Paris polyphylla callus: fibrous roots from aseptic Paris polyphylla seedlings obtained in S2 were inoculated into Paris polyphylla callus induction medium and cultured in the dark for 30 days at 22℃. The Paris polyphylla callus induction medium was a modified MS solid medium supplemented 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. After 30 days, the induction rate of Paris polyphylla callus was 61.8%.

[0075] S4. Proliferation and differentiation of adventitious roots of *Paris polyphylla*: *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℃. The *Paris polyphylla* adventitious root proliferation and differentiation medium was a modified MS solid medium supplemented with 1 mg / L 2,4-D, 3 mg / L IBA, 0.2 mg / L NAA, 0.1 mg / L KT, and 0.05 mg / L BR. After 45 days, the root proliferation coefficient of *Paris polyphylla* was 2.4.

[0076] The above-mentioned modified MS solid medium: Based on MS solid medium, NH3 is added + and NO3 - The mass molar ratio was adjusted to 1:4, the total nitrogen source concentration was adjusted to 60 mmol / L, and PO42- 3- The concentration was adjusted to 20 mmol / L, Mn 2+ Adjust the concentration to 0.1 mmol / L, Zn + Adjust the concentration to 0.01 mmol / L, BO3 3- Adjust the concentration to 0.01 mmol / L, Co 2+ Adjust the concentration to 0.01 mmol / L.

[0077] like Figures 1-3 These are sterile seedlings, callus, and adventitious roots of Paris polyphylla cultured by the method of this invention. There were no previous research reports or actual images of the callus and adventitious roots. Figure 1 The high-vitality sterile seedlings obtained in Example 2 were cultured in a sterile seedling culture medium of Paris polyphylla var. yunnanensis. The leaves were spread out and green, and the stems were upright. Figure 2 The callus tissue selected in Example 2 is induced by fibrous roots. It is white with a yellowish tinge and has a relatively hard surface texture. Figure 3 These are adventitious roots from callus differentiation in Example 2, and the transparent root tips indicate newly formed adventitious roots.

[0078] Example 3 Inducing adventitious roots of Paris polyphylla

[0079] A method for inducing adventitious roots in Paris polyphylla includes the following steps:

[0080] S1. Establishment of a sterile system for *Paris polyphylla*: Stem segments with buds were cut from *Paris polyphylla*, and sterilized sequentially with 75% alcohol (50 s) and sodium hypochlorite (20 min) before being inoculated into a sterile culture medium and cultured in the dark for 14 days at 23℃. The sterile seedling culture medium was a modified MS solid medium supplemented with 0.05 mg / L penicillin and 0.05 mg / L cephalexin. After 14 days, the contamination rate of the *Paris polyphylla* explants was 8.4%, and the survival rate was 65.8%.

[0081] S2. Preparation of aseptic seedlings of *Paris polyphylla*: The *Paris polyphylla* explants obtained in S1 were transferred to a *Paris polyphylla* aseptic seedling culture medium and cultured under light for 30 days. The *Paris polyphylla* aseptic seedling culture medium was a modified MS solid medium supplemented with 2 mg / L 6-BA, 0.5 mg / L IAA, 0.2 mg / L KT, and 0.1 mg / L ZT. The light culture conditions were: light intensity 1400 lx, light duration 12 h / d, temperature 25℃ during light exposure, and temperature 23℃ during darkness. After 30 days, complete *Paris polyphylla* aseptic seedlings were formed, yielding highly vigorous *Paris polyphylla* aseptic seedlings.

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

[0083] S4. Proliferation and differentiation of adventitious roots of *Paris polyphylla*: *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℃. The *Paris polyphylla* adventitious root proliferation and differentiation medium was a modified MS solid medium supplemented with 1 mg / L 2,4-D, 4 mg / L IBA, 0.5 mg / L NAA, 0.1 mg / L KT, and 0.1 mg / L BR. After 45 days, the root proliferation coefficient of *Paris polyphylla* was 3.1.

[0084] The above-mentioned modified MS solid medium: Based on MS solid medium, NH3 is added + and NO3 - The mass molar ratio was adjusted to 1:5, the total nitrogen source concentration was adjusted to 65 mmol / L, and PO42- 3- The concentration was adjusted to 20 mmol / L, Mn 2+ Adjust the concentration to 0.3 mmol / L, Zn + Adjust the concentration to 0.1 mmol / L, BO3 3- Adjust the concentration to 0.05 mmol / L, Co 2+ Adjust the concentration to 0.05 mmol / L.

[0085] Example 4 Inducing adventitious roots of Paris polyphylla

[0086] S1. Establishment of a sterile system for *Paris polyphylla*: Stem segments with buds were cut from *Paris polyphylla* and sterilized sequentially with 75% alcohol (50 s), mercuric chloride (10 min), and sodium hypochlorite (20 min) before being inoculated into a sterile culture medium and cultured in the dark for 14 days at 23℃. The sterile seedling culture medium was a modified MS solid medium supplemented with 0.1 mg / L penicillin and 0.02 mg / L cephalexin. After 14 days, the contamination rate of the *Paris polyphylla* explants was 9.26%, and the survival rate was 63.4%.

[0087] S2. Preparation of aseptic seedlings of *Paris polyphylla*: The *Paris polyphylla* explants obtained in S1 were transferred to a *Paris polyphylla* aseptic seedling culture medium and cultured under light for 30 days. The *Paris polyphylla* aseptic seedling culture medium was a modified MS solid medium supplemented with 3 mg / L 6-BA, 1.0 mg / L IAA, 0.5 mg / L KT, and 0.2 mg / L ZT. The light culture conditions were: light intensity 1600 lx, light duration 12 h / d, temperature 26℃ during light exposure, and temperature 24℃ during darkness. After 30 days, complete *Paris polyphylla* aseptic seedlings were formed, yielding highly vigorous *Paris polyphylla* aseptic seedlings.

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

[0089] S4. Proliferation and differentiation of adventitious roots of *Paris polyphylla*: *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℃. The *Paris polyphylla* adventitious root proliferation and differentiation medium was a modified MS solid medium supplemented with 2 mg / L 2,4-D, 5 mg / L IBA, 0.5 mg / L NAA, 0.2 mg / L KT, and 0.2 mg / L BR. After 45 days, the root proliferation coefficient of *Paris polyphylla* was 2.8.

[0090] The above-mentioned modified MS solid medium: Based on MS solid medium, NH3 is added + and NO3 - The mass molar ratio was adjusted to 1:6, the total nitrogen source concentration was adjusted to 70 mmol / L, and PO42- 3- The concentration was adjusted to 25 mmol / L, Mn 2+ Adjust the concentration to 0.5 mmol / L, Zn + Adjust the concentration to 0.2 mmol / L, BO3 3- Adjust the concentration to 0.1 mmol / L, Co 2+Adjust the concentration to 0.1 mmol / L.

[0091] Table 4. Effects of sterile culture medium on the viability of *Paris polyphylla* seedlings.

[0092] Implementation Cases Emergence time Emergence rate Emergence status Blank control (MS) — — No significant changes Example 1 26d 64.3％ A single upright stem grows Example 2 24d 69.6％ It produces upright stems, and a few produce clusters of upright stems. Example 3 27d 65.7％ It produces upright stems, and in very few cases, it produces clusters of upright stems.

[0093] As shown in Table 4, the blank control culture process used MS solid medium instead of the Paris polyphylla sterile seedling medium. All other processes and conditions were the same as steps S1-S2 in Example 2. Observations showed no significant changes in the Paris polyphylla explants before and after culture, indicating that the MS solid medium could not induce the explants to grow and emerge. The time taken for the Paris polyphylla explants in the Paris polyphylla sterile seedling medium for Examples 1-3 to emerge was recorded, and the emergence rate was calculated. The experimental results show that highly vigorous sterile seedlings can be obtained through culture using the Paris polyphylla sterile seedling medium provided by this invention, with emergence rates exceeding 64% in Examples 1-3.

[0094] Comparative Example 1

[0095] The culture of Paris polyphylla explants included the following steps: stem segments with buds were cut from Paris polyphylla, and then disinfected sequentially with 75% alcohol (50 s) and mercuric chloride (20 min) before being inoculated onto MS solid medium and cultured in the dark for 14 days at a temperature of 22-24℃. The survival rate of the Paris polyphylla explants obtained after 14 days was 0%.

[0096] Comparative Example 2

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

[0098] S1. Establishment of a sterile system for *Paris polyphylla*: Multiple stem segments with buds were collected from *Paris polyphylla*, and sterilized sequentially with 75% alcohol (50 s) and mercuric chloride (20 min) before being inoculated into a sterile culture medium and cultured for 14 days. The sterile seedling culture medium was a modified MS solid medium supplemented with 0.02 mg / L penicillin and 0.1 mg / L cephalexin. After 14 days, the survival rate of the *Paris polyphylla* explants was 62.7%.

[0099] S2. Preparation of sterile Paris polyphylla seedlings: The Paris polyphylla explants obtained in S1 were transferred to modified MS solid medium and cultured under light for 1–3 months. The light culture conditions were: light intensity 1200 lx, light duration 12 h / d, temperature 24–26℃ during light exposure, and temperature 22–24℃ during darkness. Experimental observations showed that after several months, the sterile Paris polyphylla seedlings entered a dormant and inhibited state, unable to grow further.

[0100] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for inducing adventitious roots in Paris polyphylla, characterized in that, Includes the following steps: S1. Establishment of a sterile system for Paris polyphylla: Bud-bearing stem segments of Paris polyphylla were inoculated into a sterile culture medium to obtain Paris polyphylla explants; the sterile culture medium was a modified MS solid medium with 0.02-0.1 mg / L antibiotics added. S2. Preparation of sterile Paris polyphylla seedlings: The Paris polyphylla explants obtained in S1 were transferred to a sterile Paris polyphylla seedling culture medium to obtain sterile Paris polyphylla seedlings; the sterile Paris polyphylla seedling culture medium was a modified MS solid medium supplemented 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; S3. Induction of Paris polyphylla callus: The fibrous roots of Paris polyphylla obtained from the sterile seedlings in S2 were inoculated into Paris polyphylla callus induction medium and cultured to obtain Paris polyphylla callus; the Paris polyphylla callus induction medium was a modified MS solid medium supplemented 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; S4. Proliferation and differentiation of adventitious roots of Paris polyphylla: The Paris polyphylla callus obtained in S3 was transferred to the Paris polyphylla adventitious root proliferation and differentiation medium for culture, resulting in a large number of Paris polyphylla adventitious roots; the Paris polyphylla adventitious root proliferation and differentiation medium was a modified MS solid medium supplemented 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; The modified MS solid medium is prepared by removing NH3 from the MS solid medium. + and NO3 - The mass molar ratio was adjusted to 1:(4-6), the total nitrogen source concentration was adjusted to 60-70 mmol / L, and PO4 3- The concentration was adjusted to 20-25 mmol / L, Mn 2+ The concentration was adjusted to 0.1-0.5 mmol / L, Zn + The concentration was adjusted to 0.01-0.2 mmol / L, BO3 3- The concentration was adjusted to 0.01-0.1 mmol / L, Co 2+ The concentration was adjusted to 0.01-0.1 mmol / L.

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

3. The method for inducing adventitious roots in Paris polyphylla according to claim 1, characterized in that, Steps S1, S3 and S4 involve dark incubation at a temperature of 22-24℃. The incubation time for step S1 is 2 weeks, the incubation time for step S3 is 1 month, and the incubation time for step S4 is 1.5 months.

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

5. The method for inducing adventitious roots in Paris polyphylla according to claim 1, characterized in that, In step S1, the stem segments of Paris polyphylla with buds were disinfected and then inoculated into a sterile culture medium.

6. The method for inducing adventitious roots in Paris polyphylla according to claim 5, characterized in that, The disinfection process involves sequentially or alternately disinfecting in one or more solutions of alcohol, mercuric chloride, and sodium hypochlorite. The alcohol disinfection time is 50 seconds, the mercuric chloride disinfection time is 10-20 minutes, and the sodium hypochlorite disinfection time is 10-20 minutes.

7. A combined culture medium for inducing adventitious roots of Paris polyphylla, characterized in that, The following four culture media are included: (1) A sterile culture medium for culturing Paris polyphylla explants; the sterile culture medium is a modified MS solid medium with 0.02-0.1 mg / L antibiotic added; (2) A sterile culture medium for preparing sterile Paris polyphylla seedlings; wherein the sterile culture medium is a modified MS solid medium supplemented 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; (3) A Paris polyphylla callus induction medium for inducing Paris polyphylla callus; the Paris polyphylla callus induction medium is a modified MS solid medium supplemented 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; (4) A medium for the proliferation and differentiation of adventitious roots of Paris polyphylla; the medium for the proliferation and differentiation of adventitious roots of Paris polyphylla is a modified MS solid medium supplemented 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; The modified MS solid medium is prepared by removing NH3 from the MS solid medium. + and NO3 - The mass molar ratio was adjusted to 1:(4-6), the total nitrogen source concentration was adjusted to 60-70 mmol / L, and PO4 3- The concentration was adjusted to 20-25 mmol / L, Mn 2+ The concentration was adjusted to 0.1-0.5 mmol / L, Zn + The concentration was adjusted to 0.01-0.2 mmol / L, BO3 3- The concentration was adjusted to 0.01-0.1 mmol / L, Co 2+ The concentration was adjusted to 0.01-0.1 mmol / L.

8. The combined culture medium for inducing adventitious roots of Paris polyphylla according to claim 7, characterized in that, The antibiotics are one or more of the following: penicillins, aminoglycosides, quinolones, macrolides, cephalosporins, and tetracyclines.

Images