CN113951140A

CN113951140A - Method for promoting rapid propagation of seedlings of paris polyphylla young plants

Info

Publication number: CN113951140A
Application number: CN202111333387.9A
Authority: CN
Inventors: 仇硕; 吴巧芬; 夏科; 赵志国; 蒋庆鸿
Original assignee: Guangxi Institute of Botany of CAS
Current assignee: Guangxi Institute of Botany of CAS
Priority date: 2021-11-11
Filing date: 2021-11-11
Publication date: 2022-01-21
Anticipated expiration: 2041-11-11
Also published as: CN113951140B

Abstract

The invention discloses a method for promoting the rapid propagation of seedlings of paris polyphylla young plants, which comprises breaking through seed double dormancy, pretreatment and disinfection treatment of the young plants, induction culture, multiplication culture, rooting culture and transplantation domestication. The method is adopted to breed the paris polyphylla seedlings, and the germination rate of the seeds reaches 92.5 percent; the inductivity reaches 98.33%, the culture lasts for 90 days, and the multiplication coefficient reaches 3.75; the rooting and bulb-forming rate is 96.67 percent, the roots are 3-4, the length of the roots is 4.15cm, and the diameter of the bulbs reaches 0.86 cm; the transplanting survival rate is 88.33 percent. The method can obtain a large number of excellent paris polyphylla seedlings in a short period, and the seedlings are larger in tubers and more in quantity than seedlings obtained by directly transplanting young plants outdoors. The method has the characteristics of high propagation speed, high yield, excellent seedling quality, short required time, strong practical operability and the like, can obtain a large amount of paris polyphylla seedlings in a short time, has important significance for protecting and sustainably utilizing paris polyphylla germplasm resources, and has wide application prospect.

Description

Method for promoting rapid propagation of seedlings of paris polyphylla young plants

Technical Field

The invention relates to a method for quickly propagating test-tube plantlets from young plants of Paris polyphylla, and belongs to the technical field of plant biology.

Background

Rhizoma Helminthostachydis Zeylanicae (rhizoma Helminthostachydis Zeylanicae)Paris polyphylla) Is prepared from rhizoma paridis (Liliaceae) of LiliaceaeParis) The plant is famous traditional medicinal plant, namely paris rhizome, scandent schefflera root and the like. It is suitable for mountain slope forest and irrigated cluster shady and wet forest at altitude of 1800 plus 3200 m, and has the advantages of high temperature preference, high humidity preference, high shading preference, cold resistance, drought resistance, frost resistance and sunlight resistance. Grow in sandy soil and loam with high organic matter and humus content. Paris polyphylla with multiple varieties, rhizoma paridis (rhizoma paridis)Paris polyphylla var. chinensis and Paris polyphylla (rhizoma paridis)Paris polyphylla var. yunnanensis ) Is two genuine basic species of the rhizoma paridis medicinal material specified in the 2020 edition of Chinese pharmacopoeia. With the continuous and deep research, the pharmacological efficacy of the paris polyphylla is gradually excavated, and the paris polyphylla has the efficacies of clearing away heat and toxic material, relieving swelling and pain, cooling the liver and arresting convulsion and the like, and has the effects of resisting tumor, inhibiting bacteria, stopping bleeding, regulating the immunity of the organism, resisting oxidation and the like, is a traditional Chinese medicinal material, is prepared from a plurality of Chinese patent medicines, is approved by CFDA (circulating fluid dynamics) and can be used for cosmetics,

with the continuous improvement of medicinal value, the market demand of Paris polyphylla is continuously increased, and the price is continuously increased. In order to meet the market demand and protect the wild resources of the Paris polyphylla, a plurality of researchers have conducted researches on the seed dormancy breaking technology and the tissue culture rapid propagation technology of Paris polyphylla, but no breakthrough progress is made. At present, two modes of rhizome cutting propagation and seed propagation are mainly used in artificial cultivation, and the former has the problems of high cost, easy bacterial contamination and ulceration, low propagation coefficient, even germplasm degradation and the like; the seed propagation has long dormancy period, the seedling emergence can be realized only by completing morphological after-ripening and physiological after-ripening in two winter and one summer under natural conditions, the seedling emergence rate is low, the seedling emergence is irregular, and the period from sowing to harvesting is usually 7-10 years. Therefore, the current situation that seedling resources are relatively scarce exists, and in view of the current situation, the tissue culture rapid propagation technology is always a hot research problem.

Numerous researches show that the paris polyphylla endophytic fungi are rich, the explant is difficult to sterilize thoroughly, although terminal buds, tubers, seed embryos, ovaries, buds, seedlings and the like are used for inducing callus, the problems of high pollution rate, low callus induction rate, slow proliferation, difficult differentiation and the like still exist, and the report of obtaining a large number of seedlings by using tissue culture is not available at present. Therefore, the method has the advantages that the proper explants are screened, the optimal disinfection mode is adopted, the proper induction culture medium, the proper proliferation culture medium and the proper rooting culture medium are screened, a large number of test-tube plantlets are rapidly and effectively obtained, the seedling culture time can be shortened, high-quality seedlings can be provided more rapidly, the resources of endangered medicinal plants can be protected, and the improvement of the production efficiency is promoted.

Disclosure of Invention

The invention aims to solve the defects in the prior art and provides a method for promoting the rapid propagation of seedlings of paris polyphylla young plants. The method takes the paris polyphylla with good development and application prospects as a material, cultures young plants by breaking through a seed double dormancy technology, then obtains germinated materials by sterilizing and inducing the young plants, then cuts the germinated materials for propagation culture to obtain tillered cluster buds, carries out rooting and bulb culture, after rooting, transplants and acclimates in 3-5 months or 10-11 months, screens transplanting substrates, improves transplanting survival rate and seedling quality by reasonable management of fertilization, moisture, illumination and the like, further rapidly obtains a large amount of paris polyphylla seedlings, and overcomes the problems that the conventional plant division propagation is slow, tissue culture and rapid propagation seedlings are difficult to obtain by using conventional explants.

The technical scheme for solving the technical problems is as follows:

a method for promoting the rapid propagation of seedlings of paris polyphylla young plants comprises the following steps:

step 1: obtaining of young plants of Paris polyphylla

Collecting mature uncracked rhizoma paridis capsule, taking out seed, removing red episperm, and treating with GA₃Storing the seeds in sand at low temperature for 80-90 days after seed soaking, and then placing the seeds with sand at room temperature for 60 days;

standing at room temperature, taking out seeds, soaking the seeds with GA3 again, sand-storing at low temperature for 80-90 days, and standing the seeds with sand at room temperature for 60 days to obtain germinated young plants of rhizoma paridis;

step 2: disinfection of Paris polyphylla young plants

Soaking young plants of rhizoma paridis in 0.5% detergent water for 5-10 min, cleaning surface sand, and washing with running water for at least 2 hr or soaking in 0.05% and 0.5% carbendazim for at least 2 hr;

washing with running water or soaking in carbendazim, transferring to sterile super clean bench, soaking with 75% ethanol for 45s, washing with sterile water for 1 time, and adding 0.1% HgCl₂Or soaking 5% NaClO for 6-10min for sterilization, washing with sterile water for 5 times, and drying with filter paper;

and step 3: induced culture

Inoculating the sterilized young plants obtained in the step 2 on an induction culture medium, placing the young plants in a tissue culture room for dark culture for 3-5 days, and transferring the young plants to weak light for illumination culture for 60-90 days to obtain a sprouting material;

and 4, step 4: proliferation culture

Averagely cutting the sprouting material obtained in the step (3) by using a scalpel under the aseptic condition, transferring the sprouting material into a proliferation culture medium, and continuously carrying out illumination culture for 60-90 days to obtain tillered cluster buds;

and 5: rooting and bulb-forming culture

Averagely cutting the tillering cluster buds obtained in the step (4) at a tillering point by using a scalpel, transferring the tillering cluster buds into a rooting and bulb-forming culture medium, and continuing to perform illumination culture for 60-90 days to obtain test-tube seedlings of rooting and bulb-forming stems;

step 6: transplanting domestication

And (3) moving the test-tube plantlets obtained in the step (5) out of the culture room in 3-5 months or 10-11 months, hardening the plantlets with bottles for 7-10 days, cleaning roots, airing water, planting the seedlings in a transplanting substrate in a plastic greenhouse, and transplanting and acclimating for 12-14 months to obtain the paris polyphylla seedlings.

In the method, the young plants of the Paris polyphylla in the step 1 are obtained, and GA₃The seed soaking concentration is 0-200 mg/L, the soaking time is 24h, and the low temperature is 5-8 ℃.

The induction culture medium in step 3 comprises MS or WPM, 6-BA 0-2.0mg/L, ZT 0-2.5mg/L, NAA 0.1-0.5mg/L and GA 0-0.5mg/L₃Composition, pH 5.8;

the temperature of the illumination culture is 25 +/-2 ℃, the intensity is 300-.

The proliferation culture medium in step 4 comprises MS or WPM, 6-BA 0-2.0mg/L, ZT 0-1.5mg/L, NAA 0-0.5mg/L, and GA 0-0.5mg/L₃And 0-1.0 mg/L IAA, pH 5.8;

Step 5, the rooting and bulb-forming culture medium consists of WPM, 1.0-2.0 mg/L ZT and 0.1-0.2 mg/L NAA, and the pH value is 5.8;

In the step 4, the dissecting knife is used for averagely cutting the germinated materials into 2-4 parts, and in the step 5, the dissecting knife is used for averagely cutting the germinated materials into 2-4 parts at the tillering point.

6, the transplanting domestication culture medium is a mixture of peat soil, perlite and river sand according to a volume ratio of 1:1: 1; or a mixture of bark, peat soil and sandy loam in a volume ratio of 1:1: 1; or the mixture of peat soil, yellow loam and perlite according to the volume ratio of 2:2: 1.

And 6, sterilizing the transplanting matrix by using 600 times of diluent of carbendazim or thiophanate methyl powder. The transplanting matrix is disinfected and does not contain toxic and harmful substances.

And 6, the seedling exercising is mainly performed by astigmatism, the illumination intensity is 300-.

6, spraying a foliar fertilizer for 1 time every half month and continuously spraying for 3 times after the transplanted and domesticated seedlings survive; the light intensity is 300 and 500 lux; the temperature is kept between 15 and 35 ℃ in summer, the soil is kept moist after the winter naturally overwintering, and the soil is taken out of the nursery and planted in the field in 4 to 5 months.

And 6, shading the plastic greenhouse with a 90% shading net all the year round, and spraying water by an automatic water spraying system, wherein the humidity is kept at about 80%.

The method comprises the following steps: GA₃Gibberellin (Gibberellic acid), also known as gibberellin A₃An endogenous plant growth regulator, the main functions of which in the development of plants include: stimulating cell division and elongation to promote rapid rhizome elongation; for some plants requiring stratification or light-induced germination, gibberellins can be taken off dormancy, induce mitosis and start; the germination rate of the seeds is improved; it can also participate in physiological activities such as gravimetry, tension and flowering. Low concentration of GA₃Can remarkably regulate and control the growth of plants and has high-concentration GA₃The opposite effect may occur.

6-BA, i.e., 6- (N-benzyl) aminopurine, is also known as 6-benzylaminopurine. Is the first artificially synthesized cytokinin. Has effects in inhibiting decomposition of chlorophyll, nucleic acid and protein in plant leaf, keeping green, and preventing aging; the amino acid, the auxin, the inorganic salt and the like are transported to the treatment part, and the like, and are widely used in various stages from germination to harvest of agricultural, fruit trees and horticultural crops.

ZT, zeatin, a natural cytokinin, is the 1 st natural cytokinin extracted and crystallized from grain in the filling stage of sweet corn, and has been synthesized artificially. Promoting the germination of the callus, promoting fruit setting, promoting the growth of seedling stage, delaying the yellowing of leaves and promoting the germination of some crop seeds.

NAA, i.e. 1-naphthylacetic acid. Is easy to dissolve in organic solvent, is an auxin analogue in plant growth regulators, is commonly used for producing commercial root promoting powder or root promoting agent, and is also widely applied to plant cutting propagation. It is also commonly used in plant tissue culture.

IAA, indole-3-acetic acid, is an endogenous auxin, can promote cell division and cell growth, and has a promoting effect on top bud end formation of plant twigs or buds, seedlings and the like. Inducing the formation of adventitious roots, and is also commonly used for the proliferation of tissue culture seedlings.

In the process of germinating the seeds and obtaining young plants, the GA method is adopted₃Has the function of breaking seed dormancy. In the culture medium formula of induction culture, proliferation culture and rooting culture, MS and WPM are basic culture medium, 6-BA and ZT promote cell division and growth and development, and IAA and NAA have the function of promoting root growth and development. The induction medium and the multiplication medium can greatly promote tuber development and bud development, and the induction rate is 98.33%; the rooting culture medium can greatly promote the rooting, seedling strengthening and bulb expanding development. The proliferation rate reaches 3.75 after proliferation culture for 60-90 days; in the rooting culture medium, the rooting rate is 96.67%, the number of roots is 3.84 on average, the root length is 4.15cm, and the diameter of the bulb is 0.86 cm.

In the disinfection of the young plants by the method, the explants are cleaned outdoors and then are effectively disinfected by the carbendazim pretreatment. Carbendazim is a broad-spectrum bactericide, is a high-efficiency low-toxicity systemic bactericide, and has treatment and protection effects. It has preventing and treating effect on diseases of various crops caused by fungi (such as fungi imperfecti and ascomycetes). Can be used for foliar spray, seed treatment, soil treatment and the like. Soaking the explant in 75% ethanol for 45s and 0.1% HgCl after pretreating with 0.5% carbendazim for 2 hr₂Sterilizing for 10min, controlling the pollution rate to be 1.67%, and leading the bud to grow well.

In the transplanting and domestication of the method, the optimal transplanting time is 10-11 months, and the seedlings are neat and good in quality.

The optimal transplanting substrate is a mixture of peat soil, perlite and river sand according to the mass ratio of 1:1:1, and the transplanting survival rate reaches 88.33%.

In the transplanting and domesticating of the method, 90 percent of sunshade net is needed to shade all the year round, the soil is kept moist, and the water content is more than 80 percent.

The invention has the beneficial effects that:

(1) the method of the invention utilizes GA with a certain concentration₃Storing in sand at low temperature for 80-90 days after seed soaking, and standing at room temperature for 60 days; reuse of GA₃And (4) storing the seeds in sand at a low temperature for 80-90 days after seed soaking, and placing the seeds at room temperature for 60 days to obtain germinated tender plants, wherein the germination rate reaches 92.5%, and the germination time is shortened by at least 30 days.

(2) The simple method for pretreating and disinfecting the young plants of the paris polyphylla comprises the steps of soaking the young plants in detergent for 5-10 min, cleaning sand on the surface, washing the young plants clean with tap water, and soaking the young plants in 0.5% carbendazim for 2h for pretreatment; soaking in 75% ethanol for 45s, 0.1% HgCl₂The disinfection is carried out for 10min, the pretreatment and disinfection effects are best, and the pollution rate is only 1.67%.

(3) The method comprises the steps of induction culture, multiplication culture, rooting and bulb-forming culture and the like. The method for propagating the paris polyphylla seedlings has the inductivity of 98.33 percent, the propagation coefficient of 3.75 after 90 days of culture, the rooting and bulb-forming rate of 96.67 percent, the average number of roots of 3.84 strips, the root length of 4.15cm and the bulb diameter of 0.86 cm. Can obtain a large amount of excellent test-tube plantlets in a short period, has larger tubers and more quantity than the seedlings obtained by directly transplanting young plants to the outdoor. The method has the characteristics of high propagation speed, high yield, excellent seedling quality, short required time, strong practical operability and the like, can obtain a large amount of paris polyphylla seedlings in a short time, has important significance for protecting and sustainably utilizing paris polyphylla germplasm resources, and has wide application prospect.

(4) The invention also establishes a cultivation management technical system in the rhizoma paridis transplantation domestication, which comprises transplantation time, shading, water spraying control, fertilization and the like; the optimal transplanting matrix is a mixture of peat soil, perlite and river sand according to the volume ratio of 1:1:1, and the transplanting survival rate is as high as 88.33%.

(5) The method is simple, easy to operate, wide in market prospect and suitable for large-scale popularization and application.

Drawings

FIG. 1 shows the germination of Paris polyphylla seeds of example 1;

FIG. 2 shows the germination of Paris polyphylla explants in induction medium 90d according to example 1;

FIG. 3 shows the growth of the sprouting material of Paris polyphylla in example 1 in a proliferation medium for 90 days;

FIG. 4 shows the growth of Paris polyphylla cluster buds of example 1 inoculated into rooting and bulb-forming medium for 90 days;

FIG. 5 shows the growth of Paris polyphylla cluster buds of example 1 after 150 days of inoculation into rooting and bulb-forming medium.

Detailed Description

The method of the present invention will be further described with reference to the accompanying drawings, which are provided for illustration only and are not intended to limit the scope of the invention.

Example 1

A method for quickly propagating test-tube plantlets from young plants of Paris polyphylla comprises the following steps:

step 1: obtaining of young plants of Paris polyphylla

Collecting mature uncracked rhizoma paridis capsule, taking out seed, removing red episperm, and adding 50mg/L GA₃Soaking seeds for 24h, storing in sand at 5-8 deg.C for 80d, and standing the seeds with sand at room temperature for 60 d;

after standing at room temperature, the seeds were removed and treated with 50mg/L GA again₃Soaking seeds for 24h, storing in sand at 5-8 deg.C for 80d, and standing the seeds with sand at room temperature for 60d to obtain germinated rhizoma paridis tender plant as shown in FIG. 1;

step 2: disinfection of Paris polyphylla young plants

Soaking young plants of rhizoma paridis in 0.5% detergent water for 5-10 min, cleaning sand on the surface, washing, and soaking in 0.5% carbendazim for 2 hr;

soaking with carbendazim, transferring to sterile ultraclean workbench, soaking with 75% ethanol for 45s, washing with sterile water for 1 time, and adding 0.1% HgCl₂Soaking for 10min for sterilization, washing with sterile water for 5 times, and drying with filter paper;

and step 3: induced culture

Inoculating the sterilized young plants obtained in the step 2 on an induction culture medium, placing the young plants in a tissue culture room for dark culture for 3-5 days, and then transferring the young plants to low light for illumination culture for 90 days to obtain a sprouting material, wherein the sprouting material is shown in a figure 2;

the induction culture medium consists of WPM, 2.0mg/L ZT and 0.1mg/L NAA, the pH is 5.8, and the induction rate is 98.33%;

the temperature of the illumination culture is 25 +/-2 ℃, and the illumination intensity is 300 and 500 lux; the illumination time is 12 h/d;

and 4, step 4: proliferation culture

Cutting the sprouting material obtained in the step (3) into 2-4 parts by using a scalpel on average under the aseptic condition, transferring the sprouting material into a proliferation culture medium, and continuing to perform illumination culture for 90 days to obtain tillered cluster buds, wherein the proliferation coefficient is 3.75, and is shown in figure 3;

the proliferation culture medium consists of MS, 2.0 mg/L6-BA and 1.0mg/L IAA, and the pH value is 5.8;

the temperature of the illumination culture is 25 +/-2 ℃, the intensity is 300 and 500lux, and the illumination time is 12 h/d;

and 5: rooting and bulb-forming culture

When the tillering multiple shoots obtained in the step 4 are averagely cut into 2-4 parts at the tillering point by using a scalpel, transferring the tillering multiple shoots into a rooting and bulb-forming culture medium, and continuing to perform illumination culture for 90 days to obtain test-tube plantlets of rooting and caked stems, wherein the test-tube plantlets are shown in figure 4; however, the leaf expansion requires the culture of 120-150d, and the growth condition of the culture 150d is shown in FIG. 5;

the rooting and bulb-forming culture medium consists of WPM, 0.1mg/L NAA and 2.0mg/L ZT, and the pH value is 5.8;

step 6: transplanting domestication

And (3) moving the test-tube plantlets obtained in the step (5) out of the culture room in 10-11 months, hardening the plantlets with bottles for 7-10 days, cleaning roots, airing water, planting the seedlings in a transplanting substrate in a plastic greenhouse, and transplanting and acclimating for 12-14 months to obtain the paris polyphylla seedlings.

The seedling exercising is mainly astigmatism, the illumination intensity is 300-.

The culture medium for transplanting and domesticating is a mixture of peat soil, perlite and river sand according to the volume ratio of 1:1:1, the mixture is disinfected by 600 times of diluent of carbendazim or thiophanate methyl powder before transplanting, and the transplanting survival rate reaches 88.33 percent after statistics of 30 days.

After the transplanted domesticated seedlings survive, spraying the foliar fertilizer for 1 time every half month and continuously spraying for 3 times; shading by a shading net, keeping the humidity of about 80 percent, and keeping the illumination intensity of 300 and 500 lux; the temperature is kept between 15 and 30 ℃ in summer, the soil is kept moist after the winter naturally overwintering, and the soil is taken out of the nursery and put into the field in 4 to 5 months.

Example 2

Step 1: obtaining of young plants of Paris polyphylla

GA for twice seed soaking₃The concentration of the seed soaking solution is 0mg/L, namely the control, and the seed soaking solution is used for soaking seeds for 24 hours;

the other conditions were the same as in example 1;

step 2: disinfection of Paris polyphylla young plants

Soaking young plants of rhizoma paridis in 0.5% detergent water for 5-10 min, cleaning sand on surface, transferring to ultra-clean bench, soaking in 75% ethanol for 45s, washing with sterile water for 1 time, and adding 0.1% HgCl₂Soaking for 10min for sterilization, washing with sterile water for 5 times, and drying with filter paper;

the other procedures were the same as in example 1;

and step 3: induced culture

The induction culture medium consists of MS, 1.0 mg/L6-BA and 0.25mg/L NAA, the pH value is 5.8, and the induction rate is 57.67%;

the other conditions were the same as in example 1;

and 4, step 4: proliferation culture

The proliferation culture medium consists of WPM, 1.0mg/L ZT and 0.1mg/L NAA, the pH value is 5.8, and the proliferation coefficient is 1.75;

the other conditions were the same as in example 1;

and 5: rooting and bulb-forming culture

The rooting and bulb-forming culture medium consists of WPM, 1.5mg/L ZT and 0.1mg/L NAA, and the pH value is 5.8;

the other conditions were the same as in example 1;

step 6: transplanting domestication

The transplanting matrix of the test-tube plantlet obtained in the step 5 is bark, peat soil and sandy soil in a volume ratio of 1:1:1, and other transplanting conditions are the same as those in the example 1.

Example 3

Step 1: obtaining of young plants of Paris polyphylla

GA for twice seed soaking₃The concentration of (A) is 100 mg/L;

the other conditions were the same as in example 1;

step 2: disinfection of Paris polyphylla young plants

Soaking young plants of rhizoma paridis in 0.5% detergent water for 5-10 min, cleaning sand on the surface, transferring to a clean bench, soaking in 75% ethanol for 45s, washing with sterile water for 1 time, soaking in 5% NaClO for 10min for sterilization, washing with sterile water for 5 times, and drying with filter paper;

the other procedures were the same as in example 1;

and step 3: induced culture

The induction culture medium consists of MS, 2.0 mg/L6-BA and 0.25mg/L NAA, the pH value is 5.8, and the induction rate is 70.0 percent;

the other conditions were the same as in example 1;

and 4, step 4: proliferation culture

The proliferation culture medium consists of WPM, 1.5mg/L ZT and 0.1mg/L NAA, the pH value is 5.8, and the proliferation coefficient is 2.25;

the other conditions were the same as in example 1;

and 5: rooting and bulb-forming culture

The rooting and bulb-forming culture medium consists of WPM, 1.0mg/L ZT and 0.1mg/L NAA, and the pH value is 5.8;

the other conditions were the same as in example 1;

step 6: transplanting domestication

The transplanting and domesticating culture medium is a mixture of peat soil, yellow loam and perlite in a volume ratio of 2:2:1, and other conditions are the same as in example 1.

Example 4

Step 1: obtaining of young plants of Paris polyphylla

GA for twice seed soaking₃The concentration of (A) is 200 mg/L;

the other conditions were the same as in example 1;

step 2: disinfection of Paris polyphylla young plants

Washing the explant outdoors, and then washing the explant for 2 hours by running water for pretreatment; the other steps are the same as example 1;

and step 3: induced culture

The induction medium consists of MS, 0.1mg/L of 6-BA, 0.5mg/L of NAA and 0.5mg/L of GA₃The composition, pH 5.8, inductivity 83.33%;

the other steps are the same as example 1;

and 4, step 4: proliferation culture

The proliferation culture medium consists of MS, 0.1 mg/L6-BA and 0.5mg/L NAA, the pH value is 5.8, and the proliferation coefficient is 2.33;

the other conditions were the same as in example 1;

and 5: rooting and bulb-forming culture

The rooting and bulb-forming culture medium consists of WPM, 2.0mg/L ZT and 0.2 mg/L NAA, and the pH value is 5.8;

the other conditions were the same as in example 1;

step 6: transplanting domestication

The transplanting time of the transplanting domestication is 3-5 months; the other conditions were the same as in example 1.

Example 5

Step 1: obtaining of young plants of Paris polyphylla

The procedure is as in example 1;

step 2: disinfection of Paris polyphylla young plants

After the explants are washed outdoors, 0.05 percent of carbendazim is pretreated for 2 hours; the other steps are the same as example 1;

and step 3: induced culture

The induction culture medium consists of WPM, 1.5mg/L ZT and 0.1mg/L NAA, the pH is 5.8, and the induction rate is 96.67%; the other steps are the same as example 1;

and 4, step 4: proliferation culture

The proliferation medium comprises MS, 0.1 mg/L6-BA, 0.5mg/L NAA, and 0.5mg/L GA₃Composition, pH 5.8, proliferation coefficient 3.25;

the other conditions were the same as in example 1;

and 5: rooting and bulb-forming culture

The other steps are the same as example 1;

step 6: transplanting domestication

Transplanting and domesticating time is 3-5 months, and the culture medium is a mixture of bark, peat soil and sandy loam in a volume ratio of 1:1: 1; the other steps are the same as in example 1.

Example 6

Step 1: obtaining of young plants of Paris polyphylla

The same as example 1;

step 2: disinfection of Paris polyphylla young plants

Explants were incubated with 0.1% HgCl₂Sterilizing for 6 min, and performing the same other steps as in example 1;

and step 3: induced culture

The induction culture medium consists of WPM, 2.5mg/L ZT and 0.1mg/L NAA, the pH is 5.8, and the induction rate is 91.67%; the other steps are the same as example 1;

and 4, step 4: proliferation culture

The proliferation culture medium consists of MS, 1.0 mg/L6-BA and 1.0mg/L IAA, the pH value is 5.8, and the proliferation coefficient is 2.50;

the other conditions were the same as in example 1;

and 5: rooting and bulb-forming culture

The same as example 1;

step 6: transplanting domestication

Transplanting and domesticating time is 3-5 months, and the culture medium is a mixture of peat soil, yellow loam and perlite in a volume ratio of 2:2: 1.

Examples comparative tests are as follows:

test 1: different GA₃Influence of concentration on Germination of Paris polyphylla (n ═ 3)

。

Test 2: effect of different pretreatment and Disinfection modes on Disinfection Effect

。

Experiment 3 Induction conditions of different explants of Paris polyphylla seedlings

。

Test 4: paris polyphylla cluster bud multiplication culture

。

Test 5: paris polyphylla cluster bud rooting and bulb forming culture

。

Test 6: rhizoma paridis tissue culture seedling transplantation domestication

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A method for promoting the rapid propagation of seedlings of paris polyphylla young plants is characterized by comprising the following steps:

step 1: obtaining of young plants of Paris polyphylla

Taking rhizoma paridis seed, removing seed coat, and applying GA₃Storing the seeds in sand at low temperature for 80-90 days after seed soaking, and then placing the seeds with sand at room temperature for 60 days;

standing at room temperature, taking out seed, and applying GA again₃Storing the seeds in sand at a low temperature for 80-90 days after seed soaking, and then placing the seeds with the sand at room temperature for 60 days to obtain the sprouted paris polyphylla young plants;

step 2: disinfection of Paris polyphylla young plants

Soaking young plants of rhizoma paridis in detergent water, cleaning sand on the surface, and washing with running water or with carbendazim;

washing with running water or soaking with carbendazim, transferring to sterile clean bench, and soaking in ethanolWashed with sterile water and then with HgCl₂Or NaClO sterilization, washing with sterile water after sterilization, and sucking dry surface water with filter paper after washing;

and step 3: induced culture

Inoculating the sterilized young plants obtained in the step 2 on an induction culture medium, placing the young plants in a tissue culture room for dark culture for 3-5 days, and then transferring the young plants to weak light for illumination culture for 60-90 days to obtain a sprouting material;

and 4, step 4: proliferation culture

Averagely cutting the sprouting material obtained in the step (3) under the aseptic condition, transferring the sprouting material into a proliferation culture medium, and continuing to perform illumination culture for 60-90 days to obtain tillered cluster buds;

and 5: rooting and bulb-forming culture

Cutting the tillering cluster buds obtained in the step 4 at tillering points, transferring the tillering cluster buds into a rooting and bulb-forming culture medium, and continuing to perform illumination culture for 60-90 days to obtain test-tube seedlings of rooting and bulb-forming stems;

step 6: transplanting domestication

And (3) moving the test-tube plantlets obtained in the step (5) out of a culture room in 3-5 months or 10-11 months, hardening the plantlets with bottles for 7-10 days, cleaning roots, airing water, planting the seedlings in a transplanting substrate in a greenhouse, and transplanting and acclimating the seedlings for 12-14 months to obtain the paris polyphylla seedlings.

2. The method for promoting the rapid propagation of seedlings of paris polyphylla young plants according to claim 1, which is characterized in that: step 1 obtaining of Paris polyphylla young plants, GA₃The seed soaking concentration is 0-200 mg/L, the soaking time is 24h, and the low temperature is 5-8 ℃.

3. The method for promoting the rapid propagation of seedlings of paris polyphylla young plants according to claim 1, which is characterized in that: step 2, disinfection of the paris polyphylla young plants comprises the following specific steps: soaking young plants of rhizoma paridis in 0.5% detergent water for 5-10 min, cleaning surface sand, and washing with running water for at least 2 hr or soaking in 0.05% and 0.5% carbendazim for at least 2 hr;

after washing with running water or soaking with carbendazimTransferring to a sterile superclean bench, soaking with 75% ethanol for 45s, washing with sterile water for 1 time, and adding 0.1% HgCl₂Or soaking in 5% NaClO for 6-10min for sterilization, washing with sterile water for 5 times, and drying with filter paper.

4. The method for promoting the rapid propagation of seedlings of paris polyphylla young plants according to claim 1, which is characterized in that: the induction culture medium in step 3 comprises MS or WPM, 6-BA 0-2.0mg/L, ZT 0-2.5mg/L, NAA 0.1-0.5mg/L and GA 0-0.5mg/L₃Composition, pH 5.8;

5. The method for promoting the rapid propagation of seedlings of paris polyphylla young plants according to claim 1, which is characterized in that: the proliferation culture medium in step 4 comprises MS or WPM, 6-BA 0-2.0mg/L, ZT 0-1.5mg/L, NAA 0-0.5mg/L, and GA 0-0.5mg/L₃And 0-1.0 mg/L IAA, pH 5.8;

6. The method for promoting the rapid propagation of seedlings of paris polyphylla young plants according to claim 1, which is characterized in that: step 5, the rooting and bulb-forming culture medium consists of WPM, 1.0-2.0 mg/L ZT and 0.1-0.2 mg/L NAA, and the pH value is 5.8;

7. The method for promoting the rapid propagation of seedlings of paris polyphylla young plants according to claim 1, which is characterized in that: 6, the transplanting domestication culture medium is a mixture of peat soil, perlite and river sand according to a volume ratio of 1:1: 1; or a mixture of bark, peat soil and sandy loam in a volume ratio of 1:1: 1; or the mixture of peat soil, yellow loam and perlite according to the volume ratio of 2:2: 1.

8. The method for promoting the rapid propagation of seedlings of paris polyphylla young plants according to claim 1, which is characterized in that: and 6, sterilizing the transplanting matrix by using 600 times of diluent of carbendazim or thiophanate methyl powder.

9. The method for promoting the rapid propagation of seedlings of paris polyphylla young plants according to claim 1, which is characterized in that: and 6, the seedling exercising is mainly performed by astigmatism, the illumination intensity is 300-.

10. The method for promoting the rapid propagation of seedlings of paris polyphylla young plants according to claim 1, which is characterized in that: 6, spraying a foliar fertilizer for 1 time every half month and continuously spraying for 3 times after the transplanted and domesticated seedlings survive; the illumination intensity is 300-500 lux; the temperature is kept between 15 and 35 ℃ in summer, the soil is kept moist after the winter naturally overwintering, and the soil is taken out of the nursery and planted in the field in 4 to 5 months.