Method for establishing efficient paris polyphylla tissue culture seedling system

Abstract

The invention relates to the technical field of paris polyphylla tissue culture, in particular to a method for establishing an efficient paris polyphylla tissue culture seedling system, which comprises the following steps: S1, inducing paris polyphylla secondary buds; s2, activating and budding paris polyphylla var. Yunnanensis paris buds; s3, seedling formation of paris polyphylla var. Yunnanensis; s4, pouring seedlings of paris polyphylla var. Yunnanensis; s5, repeating the steps S3-S4 for multiple times of seedling formation and seedling pouring to obtain the paris polyphylla perennial seedlings. According to the method, the problem of'false species' of the paris polyphylla is solved, waste of a large number of seedlings is prevented, an efficient paris polyphylla tissue culture system is established for the first time, perennial tissue culture seedlings in biological significance are obtained through secondary bud induction, seedling toppling and re-induction, a large number of paris polyphylla species resources are obtained, and the dilemma that paris polyphylla resources are deficient is effectively solved.

Description

Technical Field

[0001] The invention relates to the technical field of Paris polyphylla tissue culture, and more particularly to a method for establishing an efficient Paris polyphylla tissue culture seedling system. Background Art

[0002] Paris polyphylla Smith var. yunnanensis (Franch.) Hand.-Mazz. is a perennial herb of the genus Paris in the Liliaceae family. It is a variety of Paris polyphylla, also known as single-horned lotus and seven-leaf one-branch flower, and is listed as a near threatened species (NT).

[0003] Paris polyphylla is a perennial erect herb, smooth and hairless, with an erect stem 30-90cm high. The stem is single, purple-red or bluish-purple; the leaves are 4-9, oblong or elliptical-lanceolate, thick in texture, and non-membranous; the outer rows of sepals of the pedicel are green; the anthers are linear golden yellow; the fruit is spherical, often green, yellow-brown when ripe, and contains many bright red oval seeds.

[0004] Polygonum multiflorum is a shade-loving plant that often lives under shrubs. It is cold-resistant and requires high-content humus soil and an ambient temperature below 30°C. Long-term high temperatures will affect photosynthesis. In the wild, Polygonum multiflorum generally only grows one upright stem per year, and the above-ground part will fall over every year to overwinter. Most of them will bloom and bear fruit in the fourth year. During the fall period, the base of the upright stem rapidly expands and grows rapidly, and the above-ground part of the plant transfers and dies. Before the plant completely falls over, the buds for the next emergence of Polygonum multiflorum have already formed.

[0005] At present, the artificial propagation of Paris polyphylla mainly includes seed propagation and cutting propagation.

[0006] The seeds of Paris polyphylla have the characteristics of "secondary dormancy" or "double dormancy". They need to go through "two winters and one summer" before they can germinate, which takes nearly a year. Under natural conditions, it takes at least 10 years from seed germination to medicinal material harvesting. Paris polyphylla seeds begin to develop after morphological and chemical dormancy is released. The radicle breaks through the seed coat, and the epicotyl does not elongate, maintaining a dormant period of about 1 year. Paris polyphylla one- or two-year-old seedlings have only one leaf, and multiple leaves grow in the third year, but only a few bloom and bear fruit until the fourth year. The underground rhizome part of the seedlings grows in the seedling stage and enters a rapid growth period after the fourth year. Therefore, Paris polyphylla uses artificial seeds for reproduction. It takes 6-7 years from germination to its medicinal use. Compared with other medicinal plants of the same family, it has a longer growth period.

[0007] In addition to seed propagation, Paris polyphylla can also be propagated by rhizome cuttings. It is reported that asexual propagation is better than sexual propagation in terms of the yield-increasing effect of Paris polyphylla aboveground stems and rhizomes. Cutting propagation mainly involves cutting the part of the tuber with buds. Usually, the tubers are cut after the seedlings have overwintered. The cut surfaces are disinfected with wood ash. Generally, they will "turn white" after two months (that is, tiny buds will grow on the stem nodes). Plants will not be formed that year, but upright stems will grow in the spring of the following year. However, in research and actual growth, there is a phenomenon of large amounts of rotting in Paris polyphylla cut sections. Figure 2 Severe rot can be seen in the tissue culture bottles. The reason is that the endodermal cells in the stem segments of Paris polyphylla are too few to proliferate and differentiate, resulting in necrosis of plant tissue and organ cells. Summary of the invention

[0008] The present invention aims to solve the problems of the existing Paris polyphylla, such as the long growth cycle, cumbersome artificial propagation, field cultivation affected by the region and climate, large space occupation, and uneven quality of the obtained medicinal products; at the same time, it protects the Paris polyphylla species resources and meets market demand, and provides a method for establishing an efficient Paris polyphylla tissue culture seedling system, which can harvest Paris polyphylla equivalent to perennial Paris polyphylla under natural conditions within one year, with controllable quality and no heavy metal pesticide residue pollution.

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

[0010] A method for establishing an efficient Paris polyphylla tissue culture seedling system comprises the following steps:

[0011] S1. Induction of accessory buds of Paris polyphylla: taking Paris polyphylla stem segments with buds and inoculating them into Paris polyphylla accessory bud induction medium for light culture to obtain Paris polyphylla accessory buds; the Paris polyphylla accessory bud induction medium is MS solid medium with 20-30 g / L sucrose, 5-15 g / L mannose, 1-3 mg / L 6-BA, 0.05-1 mg / L 2,4-D, 0.05-0.2 mg / L GA3 and 0.2-1 mg / L ZT added;

[0012] S2. Activation of Paris polyphylla buds: The Paris polyphylla buds obtained in S1 were transferred to Paris polyphylla bud activation medium for light culture to activate buds; the Paris polyphylla bud activation medium was MS solid medium with 20-30 g / L sucrose, 1-3 mg / L 6-BA, 0.1-1 mg / L NAA, 0.1-1 mg / L IAA and 0.05-0.2 mg / L KT added;

[0013] S3. Seedlings of Paris polyphylla: The Paris polyphylla buds activated by S2 were transferred to Paris polyphylla seedling medium for light culture to obtain Paris polyphylla tissue culture seedlings; the Paris polyphylla seedling medium was MS solid medium with 20-30 g / L sucrose, 2-3 mg / L 6-BA, 0.01-0.2 mg / L NAA, 0.01-0.5 mg / L IAA and 0.05-0.2 mg / L KT added;

[0014] S4. Polygonum multiflorum seedlings: The Polygonum multiflorum tissue culture seedlings obtained in S3 were transferred to the Polygonum multiflorum seedlings medium for dark culture to obtain the Polygonum multiflorum seedlings after seedlings; the Polygonum multiflorum seedlings medium was MS solid medium with 10-20g / L sucrose, 10-20g / L glucose, 0.5-2mg / L 6-BA, 1-2mg / L ABA, 0.2-1mg / L NAA, 1-2mg / L IAA and 0.2-1mg / L KT;

[0015] S5. Perennial seedlings of Paris polyphylla: Repeat steps S3 to S4 for multiple cycles of seedling formation and seedling removal to obtain perennial tissue culture seedlings of Paris polyphylla.

[0016] In the preferred embodiment, the Paris polyphylla with buds is disinfected before inoculation in step S1, wherein the Paris polyphylla with buds is soaked in 75% alcohol for 45-60 seconds, in 0.1% HgCl2 for 15-20 minutes, and then rinsed with sterile water for 3-5 times.

[0017] In a preferred embodiment, the conditions for light culture in steps S1 to S3 are light intensity of 1200-1600 lx, light duration of 16 h / d, temperature of 24-26°C during light, and temperature of 22-24°C in darkness.

[0018] In a preferred embodiment, the dark culture temperature in step S4 is 22-24°C.

[0019] In a preferred embodiment, the culture time of steps S1 to S2 is 30-35 days.

[0020] In a preferred embodiment, the culture time of step S3 is 55-60 days.

[0021] In a preferred embodiment, the culture time of step S4 is 30-35 days.

[0022] In a preferred embodiment, in step S5, steps S3 to S4 are repeated 1 to 4 times.

[0023] Beneficial Effects of the Invention

[0024] 1. The present invention uses a polyphylla yunnanensis accessory bud induction culture medium, which solves the problem of "false seeds" during polyphylla yunnanensis planting and effectively prevents the waste of a large number of seedlings.

[0025] 2. The secondary buds induced by the present invention are secondary buds formed on the bud-bearing stem segments of Paris polyphylla, which are discovered for the first time in the research of Paris polyphylla.

[0026] 3. The present invention establishes for the first time an efficient tissue culture system for Paris polyphylla, and obtains biologically perennial tissue culture seedlings through secondary bud induction, seedling inversion and re-induction.

[0027] 4. The present invention uses the Paris polyphylla tissue culture seedling system to obtain a large amount of Paris polyphylla species resources, which can effectively solve the problem of Paris polyphylla resource shortage. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a diagram showing the raw materials and finished products at each culture stage of Example 1 of the present invention;

[0029] Figure 2 This is a picture of cell necrosis in Paris polyphylla tissues and organs.

[0030] in, Figure 1 From left to right: stem segment with buds - induction of secondary buds - activation of secondary buds to sprout - seedling formation - seedling fall. DETAILED DESCRIPTION

[0031] 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.

[0032] The invention provides a method for establishing an efficient tissue culture seedling system of Paris polyphylla for protecting Paris polyphylla species resources and solving the problems of long growth period and large market demand of Paris polyphylla.

[0033] Detailed Overview of the Invention

[0034] In the experimental process of the present invention, the stem segments with buds of Paris polyphylla were collected in Yunnan. Other materials can be obtained through commercial channels unless otherwise specified. The experimental operations involved in the present invention are carried out using conventional experimental operations and experimental conditions unless otherwise specified.

[0035] A method for establishing an efficient Paris polyphylla tissue culture seedling system comprises the following steps:

[0036] S1. Induction of accessory shoots of Paris polyphylla;

[0037] S2. Activation of budding of accessory buds of Paris polyphylla;

[0038] S3. Seedlings of Paris polyphylla;

[0039] S4. The fallen seedlings of Paris polyphylla;

[0040] S5. Perennial seedlings of Paris polyphylla.

[0041] Specifically, a method for establishing an efficient Paris polyphylla tissue culture seedling system comprises the following steps:

[0042] S1. Induction of accessory buds of Paris polyphylla: Paris polyphylla with buds was used as explants, and the Paris polyphylla with buds was sterilized and inoculated into Paris polyphylla accessory bud induction medium for light culture for 30-35 days. Paris polyphylla accessory bud induction medium is MS solid medium with 20-30g / L sucrose, 5-15g / L mannose, 1-3mg / L 6-BA, 0.05-1mg / L 2,4-D, 0.05-0.2mg / L GA3, and 0.2-1mg / L ZT added.

[0043] S2. Activation of Paris polyphylla accessory buds: Paris polyphylla accessory buds obtained in S1 were inoculated into Paris polyphylla accessory bud activation medium and cultured in the light for 30-35 days. Paris polyphylla accessory bud activation medium is MS solid medium with 20-30 g / L sucrose, 1-3 mg / L 6-BA, 0.1-1 mg / L NAA, 0.1-1 mg / L IAA and 0.05-0.2 mg / L KT added.

[0044] S3. Seedlings of Paris polyphylla: The Paris polyphylla buds activated by S2 were transferred to Paris polyphylla seedling medium for light culture for 55-60 days. The Paris polyphylla seedling medium was MS solid medium with 20-30 g / L sucrose, 2-3 mg / L 6-BA, 0.01-0.2 mg / L NAA, 0.01-0.5 mg / L IAA and 0.05-0.2 mg / L KT added.

[0045] S4. Dendrobium officinale seedlings: The Dendrobium officinale seedlings obtained in S3 were transferred to Dendrobium officinale seedling medium and cultured in the dark for 30-35 days. Dendrobium officinale seedling medium is MS solid medium with 10-20g / L sucrose, 10-20g / L glucose, 0.5-2mg / L 6-BA, 1-2mg / L ABA, 0.2-1mg / L NAA, 1-2mg / L IAA, 0.2-1mg / L KT added.

[0046] S5. Perennial seedlings of Paris polyphylla: The Paris polyphylla tissue culture seedlings after S4 seedlings were inoculated into Paris polyphylla seedling medium of S3 for light culture for 55-60 days, and then transferred to Paris polyphylla seedlings seedlings seedlings medium of S4 for dark culture for 30-35 days. Repeat this step of seedlings-seedlings process for 3 cycles to harvest perennial seedlings.

[0047] The conditions for light culture in steps S1 to S3 and S5 are preferably light intensity of 1200-1600 lx, light duration of 16 h / d, temperature of 24-26° C. during light, and temperature of 22-24° C. during darkness.

[0048] The temperature of dark culture in steps S4 to S5 is preferably 22-24°C.

[0049] In step S1, the disinfection treatment is preferably performed by soaking the Paris polyphylla stem segments with buds in 75% alcohol for 45-60 seconds, in 0.1% HgCl2 for 15-20 minutes, and rinsing with sterile water for 3-5 times.

[0050] The current 1-year-old and 5-year-old seedlings were tested for total saponins of Paris polyphylla (Parifolium saponin I (C 44 H 70 O 16 )、Parifruticosaponin II (C 51 H 82 O 20 ) and Paris polyphylla saponin VII (C 51 H 82 O 21 ) total amount) was determined by performing three experiments and taking the average values, and the contents were 0.446±0.017%, 0.737±0.021%, 0.433±0.025% and 0.768±0.015%, respectively. According to the above total saponin content of Paris polyphylla, the cultivation effects of 100-day-old seedlings and the existing 1-year-old seedlings, 2-year-old seedlings and existing 5-year-old seedlings are the same, and the 2-year-old seedlings meet the quality indicators of the Chinese Pharmacopoeia.

[0051] The experimental results show that by using the culture system established by the method of the present invention, two-year-old seedlings of Paris polyphylla can be obtained after more than 100 days of culture in the above steps S1 to S3, and five-year-old seedlings of Paris polyphylla can be obtained after more than one year of culture in the above steps S1 to S5, which greatly shortens the culture period of Paris polyphylla.

[0052] Example 1 Establishment of tissue culture seedling system of Paris polyphylla

[0053] A method for establishing an efficient Paris polyphylla tissue culture seedling system comprises the following steps:

[0054] S1. Induction of accessory buds of Paris polyphylla: Paris polyphylla with buds was used as explants. After soaking in 75% alcohol for 45 seconds, 0.1% HgCl2 for 15 minutes and washing with sterile water for 3 times, the Paris polyphylla with buds was inoculated in Paris polyphylla accessory bud induction medium and cultured in the light for 30 days. Paris polyphylla accessory bud induction medium is MS solid medium with 20g / L sucrose, 5g / L mannose, 1mg / L 6-BA, 0.05mg / L 2,4-D, 0.05mg / L GA3 and 0.2mg / L ZT added.

[0055] S2. Activation of Paris polyphylla accessory buds: Paris polyphylla accessory buds obtained in S1 were inoculated into Paris polyphylla accessory bud activation medium and cultured in the light for 30 days. Paris polyphylla accessory bud activation medium is MS solid medium with 20 g / L sucrose, 1 mg / L 6-BA, 0.1 mg / L NAA, 0.1 mg / L IAA and 0.05 mg / L KT added.

[0056] S3. Paris polyphylla seedlings: The Paris polyphylla buds activated and germinated in S2 were transferred to Paris polyphylla seedling medium and cultured in light for 55 days. The Paris polyphylla seedling medium was MS solid medium with 20 g / L sucrose, 2 mg / L 6-BA, 0.01 mg / L NAA, 0.01 mg / L IAA and 0.05 mg / L KT added.

[0057] S4. Dendrobium officinale seedlings: The Dendrobium officinale seedlings obtained in S3 were transferred to Dendrobium officinale seedling medium and cultured in the dark for 30 days. Dendrobium officinale seedling medium is MS solid medium with 10g / L sucrose, 10g / L glucose, 0.5mg / L 6-BA, 1mg / L ABA, 0.2mg / L NAA, 1mg / L IAA and 0.2mg / L KT added.

[0058] S5. Perennial seedlings of Paris polyphylla: The Paris polyphylla tissue culture seedlings obtained in S4 were inoculated into Paris polyphylla seedling medium in S3 for light culture for 55 days, and then transferred to Paris polyphylla seedling culture medium in S4 for dark culture for 30 days. Repeat this step of seedling-seedling process for 3 cycles to harvest perennial seedlings.

[0059] The light culture conditions in steps S1 to S3 and S5 include a light intensity of 1200 lx, a light duration of 16 h / d, a temperature of 24° C. during light, and a temperature of 22° C. during darkness.

[0060] The dark culture conditions in steps S4-S5 include a temperature of 22°C in the dark.

[0061] Example 2 Establishment of tissue culture seedling system of Paris polyphylla

[0062] A method for establishing an efficient Paris polyphylla tissue culture seedling system comprises the following steps:

[0063] S1. Induction of accessory buds of Paris polyphylla: Paris polyphylla with buds was used as explants. After being soaked in 75% alcohol for 45 seconds, 0.1% HgCl2 for 18 minutes and rinsed with sterile water for 4 times, the Paris polyphylla with buds was inoculated in Paris polyphylla accessory bud induction medium and cultured in the light for 32 days. Paris polyphylla accessory bud induction medium is MS solid medium with 25g / L sucrose, 10g / L mannose, 2mg / L 6-BA, 0.5mg / L 2,4-D, 0.1mg / L GA3 and 0.5mg / L ZT added.

[0064] S2. Activation of Paris polyphylla accessory buds: Paris polyphylla accessory buds obtained in S1 were inoculated into Paris polyphylla accessory bud activation medium and cultured in light for 32 days. Paris polyphylla accessory bud activation medium is MS solid medium with 25 g / L sucrose, 2 mg / L 6-BA, 0.5 mg / L NAA, 0.5 mg / L IAA, and 0.1 mg / L KT added.

[0065] S3. Paris polyphylla seedlings: The Paris polyphylla buds activated and germinated in S2 were transferred to Paris polyphylla seedling medium and cultured in light for 57 days. The Paris polyphylla seedling medium was MS solid medium with 25g / L sucrose, 2.5mg / L 6-BA, 0.1mg / L NAA, 0.2mg / L IAA, and 0.1mg / L KT added.

[0066] S4. Dendrobium officinale seedlings: The Dendrobium officinale seedlings obtained in S3 were transferred to Dendrobium officinale seedling medium and cultured in the dark for 32 days. Dendrobium officinale seedling medium is MS solid medium with 15g / L sucrose, 15g / L glucose, 1mg / L 6-BA, 1.5mg / L ABA, 0.5mg / L NAA, 1.5mg / L IAA and 0.5mg / L KT added.

[0067] S5. Perennial seedlings of Paris polyphylla: The Paris polyphylla tissue culture seedlings obtained in S4 were inoculated into Paris polyphylla seedling medium in S3 for light culture for 57 days, and then transferred to Paris polyphylla seedling culture medium in S4 for dark culture for 32 days. Repeat this step of seedling formation-seedling process for 3 cycles to harvest perennial seedlings.

[0068] The light culture conditions in steps S1 to S3 and S5 include a light intensity of 1400 lx, a light duration of 16 h / d, a temperature of 25° C. during light, and a temperature of 23° C. during darkness.

[0069] The dark culture conditions in steps S4-S5 include a temperature of 23°C in the dark.

[0070] Example 3 Establishment of tissue culture seedling system of Paris polyphylla

[0071] A method for establishing an efficient Paris polyphylla tissue culture seedling system comprises the following steps:

[0072] S1. Induction of accessory buds of Paris polyphylla: Paris polyphylla with buds was used as explants. After being soaked in 75% alcohol for 60 seconds, 0.1% HgCl2 for 20 minutes and rinsed with sterile water for 5 times, the Paris polyphylla with buds was inoculated in Paris polyphylla accessory bud induction medium and cultured in the light for 35 days. Paris polyphylla accessory bud induction medium is MS solid medium with 30g / L sucrose, 15g / L mannose, 3mg / L 6-BA, 1mg / L 2,4-D, 0.2mg / L GA3 and 1mg / L ZT added.

[0073] S2. Activation of Paris polyphylla accessory buds: Paris polyphylla accessory buds obtained in S1 were inoculated into Paris polyphylla accessory bud activation medium and cultured in the light for 35 days. Paris polyphylla accessory bud activation medium is MS solid medium with 30 g / L sucrose, 3 mg / L 6-BA, 1 mg / L NAA, 1 mg / L IAA and 0.2 mg / L KT added.

[0074] S3. Paris polyphylla seedlings: The Paris polyphylla secondary buds activated by germination obtained in S2 were transferred to Paris polyphylla seedling medium for light culture for 60 days. The Paris polyphylla seedling medium was MS solid medium with 30 g / L sucrose, 3 mg / L 6-BA, 0.2 mg / L NAA, 0.5 mg / L IAA and 0.2 mg / L KT added.

[0075] S4. Dendrobium officinale: The Dendrobium officinale tissue culture seedlings obtained in S3 were transferred to Dendrobium officinale dendrobium dendrobium culture medium and cultured in the dark for 35 days. Dendrobium officinale dendrobium culture medium is MS solid medium with 20g / L sucrose, 20g / L glucose, 2mg / L 6-BA, 2mg / L ABA, 1mg / L NAA, 2mg / L IAA and 1mg / L KT added.

[0076] S5. Perennial seedlings of Paris polyphylla: The Paris polyphylla tissue culture seedlings obtained in S4 were inoculated into Paris polyphylla seedling medium in S3 for 60 days of light culture, and then transferred to Paris polyphylla seedling culture medium in S4 for 35 days of dark culture. Repeat this step of seedling formation-seedling process for 3 cycles to harvest perennial seedlings.

[0077] The light culture conditions in steps S1 to S3 and S5 include a light intensity of 1600 lx, a light duration of 16 h / d, a temperature of 26° C. during light, and a temperature of 24° C. during darkness.

[0078] The dark culture conditions in steps S4 to S5 include a dark temperature of 24°C.

[0079] The induction rate, secondary bud activation rate and total saponin content of the Paris polyphylla tissue culture seedlings obtained in Examples 1-3 were tested, and the results are shown in Table 1.

[0080] Table 1: Subsidiary bud induction rate, subsidiary bud activation rate and total saponin content of tissue culture seedlings of Paris polyphylla in Example 1-3

[0081]

[0082] Comparative Example 1

[0083] Three two-year-old seedlings of Paris polyphylla (plants 1-3) and three five-year-old seedlings of Paris polyphylla (plants 4-6) that grew well under natural conditions were selected to test their total saponin contents. The test results are shown in Table 2.

[0084] Table 2 Total saponin content of two-year-old and five-year-old seedlings under natural conditions

[0085] Paris polyphylla Total saponin content Paris polyphylla Total saponin content Plant 1 0.447％ Plant 4 0.763％ Plant 2 0.443％ Plant 5 0.769％ Plant 3 0.441％ Plant 6 0.764％

[0086] As shown in Table 1-2, the total saponin content of the 100-day seedling of Paris polyphylla represents the total saponin content of the Paris polyphylla tissue culture seedlings detected after culturing in steps S1-S3 of Example 1-3, and the total saponin content of the one-year-old Paris polyphylla represents the total saponin content of the Paris polyphylla tissue culture seedlings detected after culturing in steps S1-S5 of Example 1-3. The experimental results show that the induction rate of the secondary buds of the present invention is as high as 76% or more, and the activation rate of the secondary buds can reach more than 65%; the total saponin content of the 100-day seedlings obtained by the present invention is similar to that of the two-year-old seedlings under natural conditions, and the total saponin content of the one-year-old seedlings is similar to that of the five-year-old seedlings under natural conditions. The culture system established by the method provided by the present invention can obtain the existing Paris polyphylla two-year-old seedlings by culturing for more than 100 days, and the existing Paris polyphylla five-year-old seedlings can be obtained in 1 year.

[0087] Comparative Example 2

[0088] The Paris polyphylla tissue pieces were inoculated into MS basic solid culture medium and cultured under light for 20 days. The light culture conditions included light intensity of 1600 lx, light duration of 16 h / d, temperature of 26°C during light and temperature of 24°C in darkness, and a large amount of rotten Paris polyphylla bud tissue and root tissue were obtained.

[0089] like Figure 2 The figure shows the bud tissue of Paris polyphylla obtained by culture using the method of Comparative Example 2 ( Figure 2 Left) and root tissue ( Figure 2 The decay image (right) shows that the proliferation of Paris polyphylla is limited during tissue culture. Figure 1 Shown are the raw materials and finished products of each stage of culture in Example 1 of the present invention. The tissue culture seedling path technology of the present invention effectively solves the problem of limited proliferation during tissue culture, and successfully obtains perennial seedlings of Paris polyphylla.

[0090] 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 establishing an efficient tissue culture seedling system of Paris polyphylla, characterized in that: The steps include: S1. Induction of Paris polyphylla side shoots: taking Paris polyphylla stem segments with buds and inoculating them into Paris polyphylla side shoot induction medium for light culture to obtain Paris polyphylla side shoots; the Paris polyphylla side shoot induction medium is MS solid medium with 20-30 g / L sucrose, 5-15 g / L mannose, 1-3 mg / L 6-BA, 0.05-1 mg / L 2,4-D, 0.05-0.2 mg / L GA3 and 0.2-1 mg / L ZT added; S2. Activation of Paris polyphylla buds: The Paris polyphylla buds obtained in S1 were transferred to Paris polyphylla bud activation medium for light culture to activate buds; the Paris polyphylla bud activation medium was MS solid medium with 20-30 g / L sucrose, 1-3 mg / L 6-BA, 0.1-1 mg / L NAA, 0.1-1 mg / L IAA and 0.05-0.2 mg / L KT added; S3. Seedlings of Paris polyphylla: The Paris polyphylla buds activated by S2 were transferred to Paris polyphylla seedling medium for light culture to obtain Paris polyphylla tissue culture seedlings; the Paris polyphylla seedling medium was MS solid medium with 20-30 g / L sucrose, 2-3 mg / L 6-BA, 0.01-0.2 mg / L NAA, 0.01-0.5 mg / L IAA and 0.05-0.2 mg / L KT added; S4. Polygonum multiflorum seedlings: The Polygonum multiflorum tissue culture seedlings obtained in S3 were transferred to the Polygonum multiflorum seedlings medium for dark culture to obtain the Polygonum multiflorum seedlings after seedlings; the Polygonum multiflorum seedlings medium was MS solid medium with 10-20g / L sucrose, 10-20g / L glucose, 0.5-2mg / L 6-BA, 1-2mg / L ABA, 0.2-1mg / L NAA, 1-2mg / L IAA and 0.2-1mg / L KT; S5. Perennial seedlings of Paris polyphylla: Repeat steps S3 to S4 for multiple cycles of seedling formation and seedling removal to obtain perennial tissue culture seedlings of Paris polyphylla.

2. The method for establishing an efficient tissue culture seedling system of Paris polyphylla according to claim 1, characterized in that: Step S1: Before inoculation, the Paris polyphylla stem segments with buds are disinfected, wherein the Paris polyphylla stem segments with buds are soaked in 75% alcohol for 45-60 seconds, soaked in 0.1% HgCl2 for 15-20 minutes, and then rinsed with sterile water for 3-5 times.

3. The method for establishing an efficient tissue culture seedling system of Paris polyphylla according to claim 1, characterized in that: The conditions of the light culture in steps S1 to S3 are as follows: light intensity of 1200-1600 lx, light duration of 16 h / d, temperature of 24-26° C. during light, and temperature of 22-24° C. during darkness.

4. The method for establishing an efficient tissue culture seedling system of Paris polyphylla according to claim 1, characterized in that: The dark culture temperature in step S4 is 22-24°C.

5. The method for establishing an efficient tissue culture seedling system of Paris polyphylla according to claim 1, characterized in that: The culture time of steps S1 to S2 is 30-35 days.

6. The method for establishing an efficient tissue culture seedling system of Paris polyphylla according to claim 1, characterized in that: The culture time of step S3 is 55-60 days.

7. The method for establishing an efficient tissue culture seedling system of Paris polyphylla according to claim 1, characterized in that: The culture time of step S4 is 30-35 days.

8. The method for establishing an efficient tissue culture seedling system of Paris polyphylla according to claim 1, characterized in that: In step S5, steps S3 to S4 are repeated 1 to 4 times.

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