A method for establishing efficient yunnan rhizoma paridis tissue culture seedling system

Abstract

The present application relates to the tissue culture technical field of Yunnan rhizoma paridis, and particularly relates to a method for establishing a high-efficiency tissue culture seedling system of Yunnan rhizoma paridis, comprising the following steps: S1. inducing Yunnan rhizoma paridis secondary buds; S2. activating the budding of Yunnan rhizoma paridis secondary buds; S3. growing Yunnan rhizoma paridis seedlings; S4. transplanting Yunnan rhizoma paridis seedlings; S5. repeating steps S3-S4 to grow and transplant seedlings for multiple times to obtain perennial seedlings of Yunnan rhizoma paridis. The present application overcomes the problem of "false seeds" of Yunnan rhizoma paridis, prevents the waste of a large number of seedlings, and first establishes a high-efficiency tissue culture system of Yunnan rhizoma paridis. The perennial tissue culture seedlings obtained through secondary bud induction, transplanting and re-induction have biological significance, a large number of Yunnan rhizoma paridis species resources are obtained, and the dilemma of the scarcity of Yunnan rhizoma paridis resources is effectively solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of Paris polyphylla Smith var. yunnanensis (Franch.) Hand.-Mazz. tissue culture, and more particularly to a method for establishing a high-efficiency Paris polyphylla Smith var. yunnanensis (Franch.) Hand.-Mazz. tissue culture seedling system. BACKGROUND

[0002] Paris polyphylla Smith var. yunnanensis (Franch.) Hand.-Mazz. is a perennial herb of the Liliaceae family and Paris genus, is one of the varieties of Paris polyphylla Smith, also known as Yiqilan and Qiyexianhua, and is listed as a near threatened species (NT).

[0003] Paris polyphylla Smith var. yunnanensis (Franch.) Hand.-Mazz. is a perennial herb, and the whole body is smooth and non-hairy. The stem is single and purple red or greenish purple. The leaf blade is long elliptic or elliptic-lanceolate, thick and non-membranous. The outer calyx lobe of the flower stalk is green. The anther is linear and golden yellow. The fruit is spherical and green, and turns yellowish brown when ripe, containing many fresh red ovate seeds.

[0004] Paris polyphylla Smith var. yunnanensis (Franch.) Hand.-Mazz. is a shade plant and often lives under shrubs. It has the characteristics of cold tolerance and needs high content of humus soil. The environmental temperature is below 30℃, and long-term high temperature will affect photosynthesis. The erect stem of wild Paris polyphylla Smith var. yunnanensis (Franch.) Hand.-Mazz. generally grows only one year. The aboveground part of Paris polyphylla Smith var. yunnanensis (Franch.) Hand.-Mazz. will be overwintered by falling off. Most of Paris polyphylla Smith var. yunnanensis (Franch.) Hand.-Mazz. will bloom and bear fruit in the fourth year. During the falling-off period, the base of the erect stem rapidly swells and grows rapidly. The material quantity of the aboveground part is transferred and dies. Before the erect stem completely falls off, the bud of the next Paris polyphylla Smith var. yunnanensis (Franch.) Hand.-Mazz. has been formed.

[0005] At present, the artificial propagation of Paris polyphylla Smith var. yunnanensis (Franch.) Hand.-Mazz. mainly includes seed propagation and cutting propagation.

[0006] The seed of Paris polyphylla Smith var. yunnanensis (Franch.) Hand.-Mazz. has the characteristics of “secondary dormancy” or “double dormancy” and needs to experience “two winters and one summer” to germinate, which lasts for nearly one year. Under natural conditions, at least 10 years are needed from seed germination to harvest of medicinal materials. The seed of Paris polyphylla Smith var. yunnanensis (Franch.) Hand.-Mazz. begins to develop after the morphological and chemical dormancy is removed. The radicle breaks through the seed coat, and the epicotyl does not elongate, which maintains the dormancy time of about one year. The one-year or two-year seedling of Paris polyphylla Smith var. yunnanensis (Franch.) Hand.-Mazz. has only one leaf. The third year, multiple leaves grow, but only a few of them bloom and bear fruit, until the fourth year. The underground rhizome part of the seedling grows in the seedling stage, and enters the rapid growth period after the fourth year. Therefore, the seed propagation of Paris polyphylla Smith var. yunnanensis (Franch.) Hand.-Mazz. needs 6-7 years from germination to harvesting for medicinal use, which is longer than other medicinal plants of the same family and genus.

[0007] In addition to seed propagation, D. yunnanensis can also be propagated by rhizome cutting. It is reported that asexual propagation is superior to sexual propagation in terms of yield increase of aboveground stems and rhizomes. Cutting propagation mainly involves cutting the rhizome with buds. The rhizome is usually cut after overwintering and seedling removal, and the cutting surface is disinfected with wood ash. Generally, the "white" (i.e., tiny bud points on the stem nodes) appears after two months, and no plants are formed this year. However, in research and actual growth, there is a phenomenon of massive rotting of D. yunnanensis segments. Figure 2 In the tissue culture bottle, severe rotting can be observed, and the reason is that there are few embryonic cells in the stem segments of D. yunnanensis, which cannot realize proliferation and differentiation, leading to cell necrosis of plant tissues and organs. SUMMARY

[0008] The present application aims to solve the problems of long growth cycle, complicated artificial propagation, large space occupation, uneven quality of medicinal material products, and the like of the existing D. yunnanensis, protect the species resources of D. yunnanensis, meet market demand, and provide a method for establishing an efficient D. yunnanensis tissue culture seedling system. The D. yunnanensis harvested within one year under natural conditions is equivalent to a perennial D. yunnanensis, and the quality is controllable without heavy metal agricultural residue pollution.

[0009] In order to achieve the above-mentioned purpose, the present application realizes the technical scheme as follows:

[0010] A method for establishing an efficient D. yunnanensis tissue culture seedling system, comprising the following steps:

[0011] S1. Induction of D. yunnanensis adventitious buds: inoculate the D. yunnanensis stem segments with buds into a D. yunnanensis adventitious bud induction medium for phototropism to obtain D. yunnanensis adventitious buds; the D. yunnanensis adventitious bud induction medium is MS solid medium added 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;

[0012] S2. Activation of D. yunnanensis adventitious bud germination: transfer the D. yunnanensis adventitious buds obtained in S1 to a D. yunnanensis adventitious bud activation medium for phototropism to activate germination; the D. yunnanensis adventitious bud activation medium is MS solid medium added 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;

[0013] S3. The seedling of Paris polyphylla var. yunnanensis: the auxiliary bud of Paris polyphylla var. yunnanensis activated in S2 is transferred to a seedling culture medium of Paris polyphylla var. yunnanensis for light culture to obtain a tissue culture seedling of Paris polyphylla var. yunnanensis; the seedling culture medium of Paris polyphylla var. yunnanensis is MS solid medium added 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;

[0014] S4. The inverted seedling of Paris polyphylla var. yunnanensis: the tissue culture seedling of Paris polyphylla var. yunnanensis obtained in S3 is transferred to an inverted seedling culture medium of Paris polyphylla var. yunnanensis for dark culture to obtain the inverted tissue culture seedling of Paris polyphylla var. yunnanensis; the inverted seedling culture medium of Paris polyphylla var. yunnanensis is MS solid medium added with 10-20 g / L sucrose, 10-20 g / L glucose, 0.5-2 mg / L 6-BA, 1-2 mg / L ABA, 0.2-1 mg / L NAA, 1-2 mg / L IAA and 0.2-1 mg / L KT;

[0015] S5. The perennial seedling of Paris polyphylla var. yunnanensis: the seedling-inverted seedling cycle of steps S3-S4 is repeated for multiple times to obtain a perennial tissue culture seedling of Paris polyphylla var. yunnanensis.

[0016] In a preferred embodiment, the stem section with buds of Paris polyphylla var. yunnanensis is subjected to sterilization treatment before inoculation in step S1, and the sterilization treatment is that the stem section with buds of Paris polyphylla var. yunnanensis is soaked in 75% alcohol for 45-60 s, 0.1% HgCl2 for 15-20 min and then rinsed with sterile water for 3-5 times.

[0017] In a preferred embodiment, the light culture in steps S1-S3 is under the condition of light intensity of 1200-1600 lx, light time of 16 h / d, temperature of 24-26 ℃ during light time and temperature of 22-24 ℃ during dark time.

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

[0019] In a preferred embodiment, the culture time in steps S1-S2 is 30-35 d.

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

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

[0022] In a preferred embodiment, in step S5, the cycle of steps S3-S4 is repeated for 1-4 times.

[0023] Advantages of the present application

[0024] 1. The present application uses the auxiliary bud induction medium of Paris polyphylla var. yunnanensis to solve the "false seed" problem of Paris polyphylla var. yunnanensis in planting, and effectively prevents the waste of a large number of seedlings.

[0025] 2. The induced side bud of the present application is the secondary bud formed on the stem section with buds of Paris polyphylla var. yunnanensis, which is first discovered in the research of Paris polyphylla var. yunnanensis.

[0026] 3. The present application first establishes a high-efficiency tissue culture system of Paris polyphylla var. yunnanensis, and the perennial tissue culture seedlings in biological sense are obtained through the induction of side buds, the uprooting of seedlings and the re-induction.

[0027] 4. The present application can obtain a large amount of resources of Paris polyphylla var. yunnanensis by using the tissue culture system of Paris polyphylla var. yunnanensis, and effectively solves the problem of the shortage of resources of Paris polyphylla var. yunnanensis. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is the display figure of raw materials and finished products at each culture stage of Example 1 of the present application;

[0029] Figure 2 is the figure of cell necrosis of Paris polyphylla var. yunnanensis.

[0030] Among them, Figure 1 From left to right, they are: stem section with buds - induction of side buds - activation of side buds - seedling formation - uprooting of seedlings. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor should belong to the protection scope of the present application.

[0032] The present application provides a method for establishing a high-efficiency tissue culture system of Paris polyphylla var. yunnanensis, in order to protect the species resources of Paris polyphylla var. yunnanensis and solve the problems of long growth period and large market demand of Paris polyphylla var. yunnanensis.

[0033] Detailed overview of the present application

[0034] In the experimental process of the present application, the stem section with buds of Paris polyphylla var. yunnanensis is collected in Yunnan, and other materials can be obtained through commercial channels if no special instructions are given. The experimental operations involved in the present application are carried out by using conventional experimental operations and experimental conditions if no special instructions are given.

[0035] A method for establishing a high-efficiency tissue culture system of Paris polyphylla var. yunnanensis, comprising the following steps:

[0036] S1. Induction of side buds of Paris polyphylla var. yunnanensis;

[0037] S2. Activation of side buds of Paris polyphylla var. yunnanensis;

[0038] S3. Seedling formation of Paris polyphylla var. yunnanensis;

[0039] S4. Uprooting of seedlings of Paris polyphylla var. yunnanensis;

[0040] S5. Perennial seedlings of Paris polyphylla var. yunnanensis.

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

[0042] S1. Induction of axillary buds of Paris polyphylla var. yunnanensis: take the stem segments with buds of Paris polyphylla var. yunnanensis as explants, disinfect the stem segments with buds of Paris polyphylla var. yunnanensis, and inoculate them into an axillary bud induction medium of Paris polyphylla var. yunnanensis for photoperated culture for 30-35 days. The axillary bud induction medium of Paris polyphylla var. yunnanensis is MS solid medium added 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.

[0043] S2. Activation of axillary buds of Paris polyphylla var. yunnanensis: inoculate the axillary buds of Paris polyphylla var. yunnanensis obtained in S1 into an axillary bud activation medium of Paris polyphylla var. yunnanensis for photoperated culture for 30-35 days. The axillary bud activation medium of Paris polyphylla var. yunnanensis is MS solid medium added 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.

[0044] S3. Seedling formation of Paris polyphylla var. yunnanensis: inoculate the activated axillary buds of Paris polyphylla var. yunnanensis obtained in S2 into a seedling formation medium of Paris polyphylla var. yunnanensis for photoperated culture for 55-60 days. The seedling formation medium of Paris polyphylla var. yunnanensis is MS solid medium added 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.

[0045] S4. Inverted seedlings of Paris polyphylla var. yunnanensis: inoculate the seedlings of Paris polyphylla var. yunnanensis obtained in S3 into an inverted seedling medium of Paris polyphylla var. yunnanensis for dark culture for 30-35 days. The inverted seedling medium of Paris polyphylla var. yunnanensis is MS solid medium added with 10-20 g / L sucrose, 10-20 g / L glucose, 0.5-2 mg / L 6-BA, 1-2 mg / L ABA, 0.2-1 mg / L NAA, 1-2 mg / L IAA, and 0.2-1 mg / L KT.

[0046] S5. Perennial seedlings of Paris polyphylla var. yunnanensis: inoculate the inverted seedlings of Paris polyphylla var. yunnanensis after S4 into the seedling formation medium of Paris polyphylla var. yunnanensis in S3 for photoperated culture for 55-60 days, and then inoculate them into the inverted seedling medium of Paris polyphylla var. yunnanensis in S4 for dark culture for 30-35 days. Repeat the seedling formation-inverted seedling process for 3 cycles to harvest the perennial seedlings.

[0047] The light culture conditions in steps S1-S3 and S5 are preferably 1200-1600lx of light intensity, 16h / d of light time, 24-26℃ of temperature during light time, and 22-24℃ of temperature during dark time.

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

[0049] The sterilization treatment in step S1 is preferably soaking the stem segments of Paris polyphylla var. yunnanensis with buds in 75% alcohol for 45-60s, 0.1% HgCl2 for 15-20min, and sterile water for 3-5 times.

[0050] The total saponins (Paris saponin I (C 44 H 70 O 16 ), Paris saponin II (C 51 H 82 O 20 ) and Paris saponin VII (C 51 H 82 O 21 ) of the existing 1-year and 5-year seedlings are determined, and each is tested for 3 times to take the average value, and the contents are 0.446±0.017%, 0.737±0.021%, 0.433±0.025% and 0.768±0.015% respectively. According to the total saponin content of Paris polyphylla var. yunnanensis above, the culture effects of the 100-day seedlings and the existing 1-year, 2-year and 5-year seedlings are the same, and the 2-year seedlings reach the quality index of Chinese Pharmacopoeia.

[0051] The experimental results show that the culture system established by the method of the present application can obtain the 2-year seedlings of Paris polyphylla var. yunnanensis after being cultured for more than 100 days by the above steps S1-S3, and can obtain the 5-year seedlings of Paris polyphylla var. yunnanensis after being cultured for more than one year by the above steps S1-S5, which greatly shortens the culture period of Paris polyphylla var. yunnanensis.

[0052] Example 1 Establishment of tissue culture system of Paris polyphylla var. yunnanensis

[0053] A method for establishing a high-efficiency tissue culture seedling system of Paris polyphylla var. yunnanensis, comprising the following steps:

[0054] S1. Induction of auxiliary buds of Paris polyphylla var. yunnanensis: taking stem segments of Paris polyphylla var. yunnanensis with buds as explants, soaking the stem segments in 75% alcohol for 45s, 0.1% HgCl2 for 15min and sterile water for 3 times respectively, and then inoculating the stem segments in an auxiliary bud induction culture medium of Paris polyphylla var. yunnanensis for light culture for 30d. The auxiliary bud induction culture medium of Paris polyphylla var. yunnanensis is MS solid medium added 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.

[0055] S2. Activating the adventitious buds of P. wilsonii: the adventitious buds of P. wilsonii obtained in S1 were inoculated on the adventitious bud activating medium of P. wilsonii and cultured under light for 30 days. The adventitious bud activating medium of P. wilsonii was MS solid medium supplemented 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.

[0056] S3. Plantlet formation of P. wilsonii: the activated adventitious buds of P. wilsonii obtained in S2 were transferred to the plantlet formation medium of P. wilsonii and cultured under light for 55 days. The plantlet formation medium of P. wilsonii was MS solid medium supplemented 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.

[0057] S4. Rooting of P. wilsonii: the plantlets of P. wilsonii obtained in S3 were transferred to the rooting medium of P. wilsonii and cultured in dark for 30 days. The rooting medium of P. wilsonii was MS solid medium supplemented with 10 g / L sucrose, 10 g / L glucose, 0.5 mg / L 6-BA, 1 mg / L ABA, 0.2 mg / L NAA, 1 mg / L IAA and 0.2 mg / L KT.

[0058] S5. Perennial plantlets of P. wilsonii: the plantlets of P. wilsonii obtained in S4 were inoculated on the plantlet formation medium of P. wilsonii in S3 and cultured under light for 55 days, and then transferred to the rooting medium of P. wilsonii in S4 and cultured in dark for 30 days. The process of plantlet formation and rooting was repeated for 3 cycles, and then the perennial plantlets were harvested.

[0059] The light culture conditions in steps S1-S3 and S5 included light intensity of 1200 lx, light duration of 16 h / d, temperature of 24℃ during light and temperature of 22℃ during dark.

[0060] The dark culture conditions in steps S4-S5 included temperature of 22℃ during dark.

[0061] Example 2 Establishment of tissue culture system of Paris polyphylla var. yunnanensis

[0062] A method for establishing an efficient tissue culture system of P. wilsonii, comprising the following steps:

[0063] S1. Inducing the adventitious buds of P. wilsonii: the stem segments with buds of P. wilsonii were used as explants, and the stem segments with buds of P. wilsonii were inoculated on the adventitious bud inducing medium of P. wilsonii and cultured under light for 32 days after being soaked in 75% alcohol for 45 s, 0.1% HgCl2 for 18 min and sterile water for 4 times. The adventitious bud inducing medium of P. wilsonii was MS solid medium supplemented with 25 g / L sucrose, 10 g / L mannose, 2 mg / L 6-BA, 0.5 mg / L 2,4-D, 0.1 mg / L GA3 and 0.5 mg / L ZT.

[0064] S2. Activation of adventitious bud of Paris polyphylla var. yunnanensis: The adventitious bud of Paris polyphylla var. yunnanensis obtained in S1 was inoculated in the activation medium of adventitious bud of Paris polyphylla var. yunnanensis and cultured under light for 32 days. The activation medium of adventitious bud of Paris polyphylla var. yunnanensis was MS solid medium added 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.

[0065] S3. Plantlet formation of Paris polyphylla var. yunnanensis: The activated adventitious bud of Paris polyphylla var. yunnanensis obtained in S2 was transferred to the plantlet formation medium of Paris polyphylla var. yunnanensis and cultured under light for 57 days. The plantlet formation medium of Paris polyphylla var. yunnanensis was MS solid medium added with 25 g / L sucrose, 2.5 mg / L 6-BA, 0.1 mg / L NAA, 0.2 mg / L IAA and 0.1 mg / L KT.

[0066] S4. Rooting of Paris polyphylla var. yunnanensis: The plantlet of Paris polyphylla var. yunnanensis obtained in S3 was transferred to the rooting medium of Paris polyphylla var. yunnanensis and cultured in dark for 32 days. The rooting medium of Paris polyphylla var. yunnanensis was MS solid medium added with 15 g / L sucrose, 15 g / L glucose, 1 mg / L 6-BA, 1.5 mg / L ABA, 0.5 mg / L NAA, 1.5 mg / L IAA and 0.5 mg / L KT.

[0067] S5. Perennial plantlet of Paris polyphylla var. yunnanensis: The plantlet of Paris polyphylla var. yunnanensis obtained in S4 was inoculated in the plantlet formation medium of Paris polyphylla var. yunnanensis in S3 and cultured under light for 57 days, and then transferred to the rooting medium of Paris polyphylla var. yunnanensis in S4 and cultured in dark for 32 days. The process of plantlet formation and rooting was repeated for 3 cycles to obtain perennial plantlet.

[0068] The light culture conditions in steps S1-S3 and S5 included light intensity of 1400 lx, light time of 16 h / d, temperature of 25 °C during light time and temperature of 23 °C during dark time.

[0069] The dark culture conditions in steps S4-S5 included temperature of 23 °C during dark time.

[0070] Example 3 Establishment of tissue culture system of Paris polyphylla var. yunnanensis

[0071] A method for establishing an efficient tissue culture system of Paris polyphylla var. yunnanensis, comprising the following steps:

[0072] S1. Induction of adventitious bud of Paris polyphylla var. yunnanensis: The stem segments with buds of Paris polyphylla var. yunnanensis were used as explants, which were soaked in 75% alcohol for 60 s, 0.1% HgCl2 for 20 min and washed with sterile water for 5 times, respectively, and then inoculated in the induction medium of adventitious bud of Paris polyphylla var. yunnanensis and cultured under light for 35 days. The induction medium of adventitious bud of Paris polyphylla var. yunnanensis was MS solid medium added with 30 g / L sucrose, 15 g / L mannose, 3 mg / L 6-BA, 1 mg / L 2,4-D, 0.2 mg / L GA3 and 1 mg / L ZT.

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

[0074] S3. Plantlet formation of Paris polyphylla var. yunnanensis: the activated secondary buds of Paris polyphylla var. yunnanensis obtained in S2 were transferred to the plantlet formation medium and cultured under light for 60 days. The plantlet formation medium was MS solid medium supplemented 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.

[0075] S4. Inverted plantlet of Paris polyphylla var. yunnanensis: the plantlets of Paris polyphylla var. yunnanensis obtained in S3 were transferred to the inverted plantlet medium and cultured in dark for 35 days. The inverted plantlet medium was MS solid medium supplemented with 20 g / L sucrose, 20 g / L glucose, 2 mg / L 6-BA, 2 mg / L ABA, 1 mg / L NAA, 2 mg / L IAA and 1 mg / L KT.

[0076] S5. Perennial plantlet of Paris polyphylla var. yunnanensis: the inverted plantlets of Paris polyphylla var. yunnanensis obtained in S4 were inoculated in the plantlet formation medium of S3 and cultured under light for 60 days, and then transferred to the inverted plantlet medium of S4 and cultured in dark for 35 days. The plantlet formation-inverted plantlet process was repeated for 3 cycles, and the perennial plantlets were harvested.

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

[0078] The dark culture conditions in steps S4-S5 included temperature of 24 °C during dark.

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

[0080] Table 1. Secondary bud induction rate, secondary bud activation rate and total saponin content of the tissue culture plantlets of Paris polyphylla var. yunnanensis in Examples 1-3

[0081]

[0082] Comparative Example 1

[0083] Three two-year-old plantlets (plants 1-3) and three five-year-old plantlets (plants 4-6) of Paris polyphylla var. yunnanensis growing in natural state were selected, and their total saponin contents were detected, and 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] Two-year-old seedling of P. polyphylla Total saponin content Five-year-old seedling of P. 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 Tables 1-2, the total saponin content of 100-day-old Paris polyphylla seedlings represents the total saponin content of Paris polyphylla tissue culture seedlings obtained after cultivation in steps S1-S3 of Examples 1-3, and the total saponin content of one-year-old Paris polyphylla seedlings represents the total saponin content of Paris polyphylla tissue culture seedlings obtained after cultivation in steps S1-S5 of Examples 1-3. Experimental results show that the lateral bud induction rate of this invention is as high as 76% or more, and the lateral bud activation rate can reach over 65%. The total saponin content of 100-day-old seedlings obtained by this invention is similar to that of two-year-old seedlings under natural conditions, and the total saponin content of one-year-old seedlings is similar to that of five-year-old seedlings under natural conditions. The existing two-year-old Paris polyphylla seedlings can be obtained after more than 100 days of cultivation using the cultivation system established by the method provided by this invention, and the existing five-year-old Paris polyphylla seedlings can be obtained in one year.

[0087] Comparative Example 2

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

[0089] like Figure 2 The image shows the Paris polyphylla bud tissue obtained by method 2 in Comparative Example 2. Figure 2 (left) and root tissue ( Figure 2 The image on the right (showing decay) indicates that *Paris polyphylla* exhibits limited proliferation during tissue culture. For example... Figure 1 The image shown is a display of the raw materials and finished products at each stage of the culture in Example 1 of the present invention. Through the tissue culture seedling pathway technology of the present invention, the problem of limited proliferation during the tissue culture process is effectively solved, and perennial seedlings of Paris polyphylla yunnanensis are successfully obtained.

[0090] 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 establishing an efficient tissue culture plantlet system of Paris polyphylla var. yunnanensis, characterized in that, Comprise the following steps: S1. Induction of the secondary bud of Paris fargesii Franch: the stem section with bud of Paris fargesii Franch is inoculated in the secondary bud induction medium of Paris fargesii Franch to carry out the light culture, and the secondary bud of Paris fargesii Franch is obtained; the secondary bud induction medium of Paris fargesii Franch is MS solid medium added 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; S2. Activation of the secondary bud of Paris fargesii Franch: the secondary bud of Paris fargesii Franch obtained in S1 is transferred to the secondary bud activation medium of Paris fargesii Franch to carry out the light culture, and the bud is activated; the secondary bud activation medium of Paris fargesii Franch is MS solid medium added 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; S3. Seedling of Paris fargesii Franch: the bud activated in S2 is transferred to the seedling medium of Paris fargesii Franch to carry out the light culture, and the tissue culture seedling of Paris fargesii Franch is obtained; the seedling medium of Paris fargesii Franch is MS solid medium added 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; S4. Inverted seedling of Paris fargesii Franch: the tissue culture seedling of Paris fargesii Franch obtained in S3 is transferred to the inverted seedling medium of Paris fargesii Franch to carry out the dark culture, and the tissue culture seedling of Paris fargesii Franch after the inverted seedling is obtained; the inverted seedling medium of Paris fargesii Franch is MS solid medium added with 10-20 g / L sucrose, 10-20 g / L glucose, 0.5-2 mg / L 6-BA, 1-2 mg / L ABA, 0.2-1 mg / L NAA, 1-2 mg / L IAA and 0.2-1 mg / L KT; S5. Perennial seedling of Paris fargesii Franch: the seedling-inverted seedling is repeated for multiple cycles, and the perennial tissue culture seedling of Paris fargesii Franch is obtained.

2. The method according to claim 1, wherein, The stem section with bud of Paris fargesii Franch before inoculation in step S1 is subjected to disinfection treatment, and the disinfection treatment is that the stem section with bud of Paris fargesii Franch is soaked in 75% alcohol for 45-60 s, 0.1% HgCl2 for 15-20 min, and then rinsed with sterile water for 3-5 times.

3. The method according to claim 1, wherein, The light culture in steps S1-S3 is under the condition of light intensity 1200-1600 lx, light time length 16 h / d, temperature 24-26 ℃ during light time, and temperature 22-24 ℃ during dark time.

4. The method according to claim 1, wherein, The dark culture temperature in step S4 is 22-24 ℃.

5. The method according to claim 1, wherein, The culture time in steps S1-S2 is 30-35 d.

6. The method according to claim 1, wherein, The culture time in step S3 is 55-60 d.

7. The method according to claim 1, wherein, The culture time in step S4 is 30-35 d.

8. The method according to claim 1, wherein, In step S5, the number of times of repeating steps S3-S4 is 1-4.

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