CN111165356B - Tissue culture propagation method of peony - Google Patents

Tissue culture propagation method of peony Download PDF

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Publication number
CN111165356B
CN111165356B CN202010129492.XA CN202010129492A CN111165356B CN 111165356 B CN111165356 B CN 111165356B CN 202010129492 A CN202010129492 A CN 202010129492A CN 111165356 B CN111165356 B CN 111165356B
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culture
culture medium
medium
propagation
peony
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CN111165356A (en
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闫晓芳
屈武斐
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Shanghai Peilin Biotechnology Co ltd
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Shanghai Peilin Biotechnology Co ltd
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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/001Culture apparatus for tissue culture
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/005Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008Methods for regeneration to complete plants

Abstract

The invention belongs to the field of biology and biotechnology, and discloses a tissue culture propagation method of peony. The method mainly comprises the following steps: primary culture, propagation and proliferation culture, strong seedling culture, rooting culture and domestication and transplantation. The method can maintain the female parent genetic character of the excellent peony variety, has high propagation coefficient, can obtain a large amount of robust tissue culture seedlings in a short period, and has developed root system and high transplanting survival rate. The invention provides a rapid propagation way which can effectively meet the requirement of large-scale industrialized seedling culture.

Description

Tissue culture propagation method of peony
Technical Field
The invention belongs to the field of biology and biotechnology, and particularly relates to a tissue culture propagation method of peony.
Background
Peony (Paeonia suffruticosa) belongs to Paeoniaceae (Paeoniaceae) Paeonia (Paeonia) woody plant, is a traditional famous flower originally produced in China, and is valued as "the Chinese celestial fragrance" and the king of the flower. Peony is an important medicinal plant and also an important woody oil crop.
The traditional propagation modes of the peony include seed propagation in sexual propagation, plant division propagation, layering propagation, grafting propagation and the like in vegetative propagation. Due to low setting rate and even weak seed of various peonies and poor seed development, excellent plants are difficult to obtain by seed propagation, and seedlings are difficult to maintain the excellent characters of female parents; the conventional asexual propagation modes such as grafting, plant division and the like are limited by the growing season, the propagation rate is low, the scale cannot be formed, the market demand cannot be met, and the propagation speed limits the industrialization and the commercialized production of the peony seedlings. The method for breeding the peony by utilizing the tissue culture technology is an effective way for improving the breeding rate of the peony and is convenient for large-scale production.
At present, a relatively complete technical system is not formed in tissue culture of peony at home and abroad, so that the peony tissue culture method cannot be used in production practice all the time. The main problems are that (1) the tissue culture propagation multiplication coefficient is low; (2) the rooting rate is low, the rooting quality is poor, and the transplanting is influenced; (3) the transplanting survival rate is low, and the death rate in the transplanting and domesticating stage is high. The extremely low survival rate of the transplanted peony roots limits the application of the tissue culture technology in practice, which is the bottleneck of realizing industrial seedling culture of the peony. Domestic reports on peony test-tube plantlets are numerous, but the peony test-tube plantlets are not really applied to production. FIG. 15 is a photograph of a part of peony rooted seedlings in the year 2019 at the national gardening fair of Beijing, which have few roots and weak seedlings and are seriously browned. FIG. 16 is a photograph of the propagated seedlings of peony in the competition at the national gardening fair of Beijing in 2019, which has low propagation and proliferation rate, weak growth, thin and small plant, few leaves and light leaf color.
Therefore, the two steps of rooting and domestication and transplantation are solved, and the important breakthrough of realizing industrial production of the peony is realized.
Disclosure of Invention
The invention aims to provide a tissue culture propagation method capable of efficiently propagating peony.
In a first aspect of the present invention, there is provided a method for inducing peony to root, the method comprising: (1) obtaining a stem section with buds of a peony explant, starting culture medium culture, and inducing to obtain adventitious buds; the start-up medium comprises: 6-benzyladenine, naphthylacetic acid, gibberellin, PVPP and silver nitrate; (2) inoculating the adventitious bud induced in the step (1) into a propagation culture medium for culture, and inducing to generate cluster buds; the propagation culture medium comprises: 6-benzyladenine, indole-3-butyric acid, hymexazol, gibberellin, PVPP, ascorbic acid; (3) inoculating the cluster buds obtained in the step (2) into a strong seedling culture medium to obtain strong seedling; wherein, the strong seedling culture medium comprises: 6-benzyladenine, naphthylacetic acid and ascorbic acid; (4) cutting single buds from the bud cluster in the step (3), and culturing by using a rooting culture medium to obtain rooted test-tube plantlets; wherein, the rooting culture medium comprises: indole-3-butyric acid, naphthylacetic acid and activated carbon.
In a second aspect of the present invention, there is provided a method for transplanting and acclimatizing peony tissue culture seedlings, comprising: (a) inducing by the method of the first aspect to obtain rooted peony test-tube plantlets; (b) transplanting the rooted test-tube plantlet in the step (a) into a seedling culture substrate for seedling hardening; (c) transplanting the acclimated seedlings of the step (b) to a natural environment.
In a preferred embodiment, in the step (1), when the medium is started to culture, the culture is performed in the dark for 3 to 4 days, and then the light culture is performed.
In another preferred example, in step (1), the culture in the starting medium is initiated for 15 to 20 days.
In another preferred example, in step (1), the start medium comprises: 6-benzyladenine 0.5-1.0 mg/l; 0.20-0.50 mg/L of naphthylacetic acid; gibberellin 0.6-1.0 mg/L; PVPP 200-; 0.5-1.0 mg/L of silver nitrate.
In another preferred embodiment, in step (2), the propagation medium comprises: 6-benzyladenine 0.50-1.50 mg/l; indole-3 butyric acid 0.10-0.30 mg/l; hymexazol 5.0-7.0 mg/L; gibberellin 0.6-1.0 mg/L; PVPP 200-; ascorbic acid 40-60 mg/l.
In another preferred example, in the step (2), the time for culturing the propagation medium is 25 to 35 days.
In another preferred example, in the step (3), the strong seedling culture medium comprises: 6-benzyladenine 0.50-1.50 mg/l; 0.10-0.30 mg/L of naphthylacetic acid; ascorbic acid 40-60 mg/l.
In another preferred example, in the step (3), the period of culturing in the strong seedling culture medium is 20 to 30 days.
In another preferred example, in the step (4), the culture is performed in a rooting medium, and after 4 to 6 days of dark culture, light culture is performed.
In another preferred example, in step (4), the rooting medium comprises: indole-3 butyric acid 1.0-3.0 mg/l; 0.5-2.0 mg/L of naphthylacetic acid; 1.0-2.0 g/L of active carbon.
In another preferred example, in step (4), the rooting medium is cultured for 30 to 40 days.
In another preferred example, in the step (b), the seedling substrate includes peat soil, perlite, vermiculite or a combination thereof.
In another preferred example, in the step (b), the transitional culture is carried out before seedling exercising, the temperature is 20-22 ℃, and the relative humidity is 80-90%.
In another preferred example, in the step (b), the relative humidity during seedling exercising is 80% or less.
In another preferred example, in the first and second aspects, the start medium, the propagation medium, the strong seedling medium, or the rooting medium uses an MS medium or an 1/2MS medium as a basal medium.
In another preferred example, the start-up medium takes 1/2MS culture medium as a basic culture medium; the propagation and proliferation culture medium takes an MS culture medium as a basic culture medium; the strong seedling culture medium takes an MS culture medium as a basic culture medium; or the rooting medium takes 1/2MS culture medium as a basic culture medium.
In another preferred embodiment, the culture medium further comprises sucrose (preferably 30-40 g/L) and agar (preferably 6-7 g/L).
In another preferred example, calcium chloride is further added into the starting culture medium, the propagation and proliferation culture medium, the strong seedling culture medium or the rooting culture medium; preferably 400-600 mg/l calcium chloride is added.
In another preferred example, before the step (1), the method further comprises sterilizing the peony explants; preferably, 2-3 times of sodium hypochlorite solution disinfection is adopted; more preferably, the explants are rinsed with a detergent solution, then sterilized with an alcohol solution, and then sterilized with 0.5% sodium hypochlorite 2-3 times.
In another preferred embodiment, each solution is sterilized and then rinsed with sterile water 1-3 times.
In another preferred example, the alcohol solution is 75% alcohol.
In another preferred embodiment, the detergent solution is 1% detergent water.
In a third aspect of the present invention, there is provided a kit for culturing peony, comprising: starting a culture medium, a propagation and proliferation culture medium, a strong seedling culture medium and a rooting culture medium; wherein the start-up medium comprises: 6-benzyladenine 0.5-1.0 mg/l; 0.20-0.50 mg/L of naphthylacetic acid; gibberellin 0.6-1.0 mg/L; PVPP 200-; 0.5-1.0 mg/L of silver nitrate; the propagation and proliferation culture medium comprises: 6-benzyladenine 0.50-1.50 mg/l; indole-3 butyric acid 0.10-0.30 mg/l; hymexazol 5.0-7.0 mg/L; gibberellin 0.6-1.0 mg/L; PVPP 200-; ascorbic acid 40-60 mg/l; the strong seedling culture medium comprises: 6-benzyladenine 0.50-1.50 mg/l; 0.10-0.30 mg/L of naphthylacetic acid; ascorbic acid 40-60 mg/l; the rooting medium comprises: indole-3 butyric acid 1.0-3.0 mg/l; 0.5-2.0 mg/L of naphthylacetic acid; 1.0-2.0 g/L of active carbon.
In another preferred embodiment, the kit further comprises a culture medium selected from the group consisting of: MS culture medium, 1/2MS culture medium, cane sugar, agar and calcium chloride.
In the fourth aspect of the invention, the application of the kit is provided, and the kit is used for inducing peony to root or culturing peony.
Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.
Drawings
FIG. 1 is a representative picture of tissue-cultured seedlings obtained after the propagation culture in the example.
FIG. 2 is a representative picture of a part of tissue-cultured seedlings obtained after the propagation culture in the example.
FIG. 3 is a photograph of the roots of a tissue-cultured rooted seedling.
FIG. 4 is a drawing of a tissue-cultured rooted seedling.
FIG. 5, transplanting of 3 months tissue culture seedlings.
FIG. 6, 6 months of tissue culture seedlings were transplanted.
FIG. 7, 1 year of tissue culture plant transplantation.
FIG. 8, 2 years of tissue culture plants are transplanted.
FIG. 9, 2-year-old tissue-cultured plants (root growth state) were transplanted.
FIG. 10, 2-year-old tissue-cultured plants (growth state of roots) were transplanted.
FIG. 11, 3 years of tissue culture plants.
FIG. 12, 3-year-old tissue culture plants (growth state of roots).
FIG. 13, 3 years of tissue culture plants (flowering stage) were transplanted.
FIG. 14, 3-year transplantation of tissue-cultured plants (fruiting stage).
FIG. 15 is a photograph of peony seedlings of the prior art, the left side view being a view from the side of the container and the right side view being a view from the bottom of the container.
FIG. 16 is a photograph of a peony tissue culture propagation seedling in the prior art.
Detailed Description
The inventor discloses a tissue culture rapid propagation method capable of efficiently propagating peony through extensive research and analysis. On the basis of utilizing totipotency of plant tissue cells and plant tissue culture technology, the method of the invention effectively optimizes start culture, propagation, seedling strengthening and rooting, optimizes transplant domestication and later-stage culture management and can efficiently obtain a large number of peony plants through deep research.
Term(s) for
As used herein, a "stem segment with buds" is a stem segment with buds on the shoots of a plant, wherein the buds can be terminal buds or axillary buds, and can be shoot buds or dormant buds.
As used herein, the term "dark culture" refers to a 24-hour day culture in a dark environment.
As used herein, the term "light culture" refers to a 24-hour day culture with 16-hour light and 8-hour dark, wherein the light intensity is 2000-.
As used herein, the "multiplication factor" is the multiple by which a plant undergoes propagation from the initiation of culture (before the induction culture of the propagation medium) to the rooting stage of the peony.
As used herein, the natural environment includes, but is not limited to: fields, nurseries, forests and other environments.
As used herein, "comprising," "including," or "containing" means that the various ingredients can be used together in a mixture, composition, or medium of the invention. Thus, the terms "consisting essentially of and" consisting of are encompassed by the terms "comprising," including, "or" including.
Tissue culture propagation method
The invention provides a method for inducing peony tissue culture, which mainly comprises the following steps: primary culture, propagation and proliferation culture, strong seedling culture, rooting culture and domestication and transplantation. In addition, preferably, before the primary culture, a step of disinfection is also included; in the test-tube seedling transplanting, the method preferably further comprises the processes of transitional culture, domestication and seedling hardening.
Primary culture
When primary culture is carried out, the current-year branches of the peony can be obtained to be used as explants, culture medium culture is started, and adventitious buds are induced.
When primary culture is performed, the start-up medium comprises: 6-benzyladenine, naphthylacetic acid, gibberellin, PVPP and silver nitrate. The ratio of the above components at different concentrations plays an extremely important role in the cultivation at this stage.
In this stage, the inventor finds that silver nitrate can promote the induction of peony buds and improve the survival rate of the buds, thereby effectively improving the success rate of primary culture.
In a preferred embodiment of the present invention, the medium culture is started by culturing in the dark for 3 to 4 days and then culturing in the light. Dark culture for the first few days is beneficial to bud sprouting, prevents browning, and then is converted into light culture, so that the explants can grow normally.
In a preferred embodiment of the present invention, the culture in the starting medium is carried out for 15 to 20 days. It is to be understood that although such a period is preferable in the present invention, appropriate changes may be made according to the need for cultivation and the difference of seasons for cultivation, and such changes are also included in the present invention.
Propagation and multiplication culture
When the propagation and multiplication culture is performed, the adventitious bud obtained as described above is induced to produce a cluster bud.
In a preferred embodiment of the present invention, stem segments with shoots are excised from adventitious shoots and inoculated onto a propagation medium to induce cluster shoots, wherein the propagation medium comprises: 6-benzyladenine, indole-3 butyric acid, hymexazol, gibberellin, PVPP and ascorbic acid.
In the propagation culture medium, the reasonable proportion of each component ensures the induction of the bud. The inventor finds that hymexazol not only can be used as a bacteriostatic agent for peony tissue culture, but also can promote propagation and proliferation. The combined application of PVPP and ascorbic acid effectively reduces browning of the culture medium and avoids poisoning of browning substances to peony seedlings.
In a preferred embodiment of the present invention, the time for culturing the propagation medium is 25 to 35 days. It is to be understood that although such time periods are preferred in the present invention, appropriate variations may be made depending on differences in different clones, and such variations are also encompassed in the present invention.
In the propagation culture medium, multiple cluster buds are induced to generate, and one cluster bud can be induced to generate multiple cluster buds, so that the propagation coefficient is improved.
Strong seedling culture
After obtaining the cluster buds, the inventor carries out strong seedling culture, which comprises culturing the obtained cluster buds with a strong seedling culture medium to obtain peony bud seedlings.
The strong seedling culture medium comprises 6-benzyladenine, naphthylacetic acid and ascorbic acid. Among them, the combined use of 6-benzyladenine and naphthylacetic acid is advantageous for elongation of the cluster buds at this stage. The use of ascorbic acid prevents browning of the medium.
In a preferred embodiment of the present invention, the culture time of the strong seedling medium is 20 to 30 days.
Rooting culture
After strong seedling culture, the invention carries out rooting culture on peony seedlings, and comprises the following steps: cutting the obtained cluster buds into single clusters, and culturing by a rooting culture medium to obtain rooted test-tube plantlets.
The rooting medium comprises indole-3-butyric acid, naphthylacetic acid and activated carbon. The reasonable proportion of each component is beneficial to the induction and the elongation of roots.
In a preferred embodiment of the present invention, the culture is performed in a rooting medium, followed by dark culture for 4 to 6 days and then light culture. Dark culture in the first few days is favorable for root induction, and then light culture is carried out, so that the overground part of the plant can grow.
The rooting stage is an important stage in the tissue culture process and determines the success or failure of the whole tissue culture. In the invention, the test-tube plantlet obtained after rooting culture is robust and has developed root system.
Test-tube plantlet transplanting
After the rooted test-tube plantlets are obtained, the inventor transplants the test-tube plantlets into a seedling culture substrate for domestication and seedling hardening; and then transplanting the trained seedlings to the natural environment.
Domestication and acclimatization may employ techniques known in the art. As a preferable mode of the invention, the seedling substrate comprises a combination of peat soil, perlite and vermiculite.
Generally, a period of high-humidity transitional culture is firstly carried out, and then low-humidity seedling exercising is carried out. As a preferable mode of the invention, the temperature of the transitional culture is 20-22 ℃, and the relative humidity is 80-90%; the relative humidity during seedling hardening is less than 80%.
Disinfection
According to the technical scheme, the branches of the peony are used as explants, so that the method further comprises the step of branch disinfection before initial culture.
The times of disinfection and the proportion of contamination in the subsequent cultivation process have a certain relationship, and the increase of the contamination rate can be caused when the disinfection is insufficient. Therefore, as a preferred mode of the present invention, explants are rinsed with a detergent solution, then sterilized with an alcohol solution, and then sterilized with 0.5% sodium hypochlorite 2-3 times. Each solution was sterilized and washed with sterile water 1-3 times.
In the above culture process of the present invention, the MS culture medium or 1/2MS culture medium is used as the basal medium. As a preferable mode of the invention, the start culture medium and the rooting culture medium take 1/2MS culture medium as basic culture medium; the propagation and proliferation culture medium and the strong seedling culture medium take an MS culture medium as a basic culture medium.
The MS culture medium or 1/2MS culture medium can be prepared by self or obtained by commercial products. It is understood that media which are appropriately modified (e.g., fine-tuned or replaced in part) on the basis of these basal media, but which retain their function as basal media, are also encompassed by the present invention.
Culture medium and kit for tissue culture propagation
On the basis of the optimization of the cultivation process, the invention also provides a kit for cultivating peony, which comprises: a starting culture medium, a propagation and proliferation culture medium, a strong seedling culture medium and a rooting culture medium.
In a preferred embodiment of the present invention, the priming medium comprises: 6-benzyladenine 0.5-1.0 mg/l; 0.20-0.50 mg/L of naphthylacetic acid; gibberellin 0.6-1.0 mg/L; PVPP 200-; 0.5-1.0 mg/L of silver nitrate; the propagation and proliferation culture medium comprises: 6-benzyladenine 0.50-1.50 mg/l; indole-3 butyric acid 0.10-0.30 mg/l; hymexazol 5.0-7.0 mg/L; gibberellin 0.6-1.0 mg/L; PVPP 200-; ascorbic acid 40-60 mg/l; the strong seedling culture medium comprises: 6-benzyladenine 0.50-1.50 mg/l; 0.10-0.30 mg/L of naphthylacetic acid; ascorbic acid 40-60 mg/l; the rooting medium comprises: indole-3 butyric acid 1.0-3.0 mg/l; 0.5-2.0 mg/L of naphthylacetic acid; 1.0-2.0 g/L of active carbon;
in a preferred mode of the invention, the kit further comprises sucrose, agar and calcium chloride for adding into the culture medium.
In a preferred embodiment of the present invention, the kit further comprises some basic culture media for plant cultivation, including: MS medium, or 1/2MS medium.
As a preferred mode of the present invention, the kit further comprises an instruction manual, wherein a method for using each culture medium is described, or a method for treating a plant or plant tissue before or after using the culture medium is also described, thereby being beneficial to guide a person skilled in the art to use the kit in a proper method.
The main beneficial technical effects of the invention comprise:
the biggest difficulty of peony tissue culture is that the rooting rate is low, and seedlings with developed root systems are difficult to obtain; the invention solves the technical problems of low rooting rate and poor rooting quality.
The invention can maintain the female parent genetic character of the excellent peony variety, the propagation coefficient is 4-6, a large amount of robust tissue culture seedlings can be obtained in a short period, the root system is developed, the transplanting survival rate can reach 90 percent, and the invention is an effective rapid propagation way which can meet the requirement of large-scale industrialized seedling.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.
Materials and methods
MS medium was purchased from PhytoTech.
1/2MS culture medium: based on MS culture medium, macroelements (inorganic salts) are added in half, and microelements and vitamins are not added in half.
The start-up medium formulation is given in table 1.
TABLE 1
The formula of the propagation medium is shown in Table 2.
TABLE 2
The formula of the strong seedling culture medium is shown in the table 3.
TABLE 3
The rooting medium formulation is shown in table 4.
TABLE 4
Unless otherwise stated, all media were autoclaved at 121 ℃ for 20 minutes with the addition of 30 g/l sucrose and 6.5 g/l agar (pH 5.8-6.0).
Unless otherwise stated, the cultivation temperature is 25 ℃. + -. 2 ℃.
The light culture is carried out for 16h/8h (day/night) and the light intensity is 2000-2500 Lux, unless otherwise specified. When the dark culture is performed, the culture is performed in the dark environment for 24 hours all day.
Example 1 tissue culture propagation of peony 1
The tissue culture propagation of the peony comprises the following steps:
(a) establishment of sterile System
Obtaining branches with plump terminal buds and axillary buds on the current-year peony plants, rinsing the branches with 1% of detergent water, washing the branches with running water for 30 minutes, disinfecting the branches with 75% of alcohol in an ultra-clean workbench for 30 seconds, and washing the branches with sterile water for 2 times; the method comprises sterilizing for 2 times, treating with 0.5% sodium hypochlorite solution for 4 min, washing with sterile water for 2 times, soaking in 0.5% sodium hypochlorite solution for 4-6 min, and washing with sterile water for 3 times.
(b) Primary culture
Cutting the sterilized branches into 2-3 cm stem segments with buds, inoculating the stem segments into a starting culture medium, carrying out dark culture for 3 days, and then transferring to normal illumination culture.
The start-up medium contains: 1/2MS +450 mg/l calcium chloride +1.0 mg/l 6-benzyladenine (6-BA) +0.2 mg/l naphthylacetic acid (NAA) +0.6 mg/l gibberellin (GA3) +400 mg/l PVPP +0.5 mg/l silver nitrate.
And (4) performing primary culture for 15-20 days, and germinating on the explant.
(c) Propagation and multiplication culture
After primary culture for 15-20 days, the induced bud is cut into 1.5-2 cm single bud and inoculated into propagation and proliferation culture medium for culture.
The propagation and proliferation culture medium contains: MS +450 mg/l calcium chloride +0.5 mg/l 6-benzyladenine (6-BA) +0.2 mg/l indole-3-butyric acid (IBA) +5 mg/l hymexazol +0.6 mg/l gibberellin +250 mg/l PVPP +40 mg/l ascorbic acid (Vc).
Propagating, proliferating and culturing for 25-30 days to induce multiple shoots, and generating 4-6 multiple shoots in one primary culture.
Representative plants obtained by propagation and proliferation culture are shown in FIG. 1.
(d) Strong seedling culture
And transferring the cluster buds induced from the propagation culture medium into a strong seedling culture medium for culture.
The strong seedling culture medium contains: MS +400 mg/l calcium chloride +0.5 mg/l 6-benzyladenine (6-BA) +0.10 mg/l naphthylacetic acid (NAA) +40 mg/l ascorbic acid (Vc).
Culturing in strong seedling culture medium for 20-30 days to obtain strong bud with height of 2-3.5 cm.
(e) Rooting culture
Cutting the peony clump buds with high growth vigor and the height of about 2.5-3 cm into single buds, selecting robust single buds with more than 3 leaves, and inoculating the strong single buds into a rooting culture medium for culture. The cells were kept in the dark for 5 days before being transferred to light culture.
The rooting culture medium comprises: 1/2MS +400 mg/l calcium chloride +1 mg/l indole-3-butyric acid (IBA) +0.5 mg/l naphthylacetic acid (NAA) +1.0 g/l Activated Carbon (AC).
Rooting culture for 20-25 days (from dark culture), wherein more than 90% of seedlings start to root, the root length is 4-6 cm in 30-40 days, and each seedling has 7-10 roots on average.
After rooting culture, the rooting of the obtained plants was observed from the bottom of the culture vessel as shown in FIG. 3.
(f) Transplanting domestication
Taking out the strong rooting seedling with root length of 4-7 cm and rooting culture period of 30-40 days from the culture room, culturing in scattered light at 20-22 deg.C in greenhouse, and gradually removing the cover.
Transplanting after 5-7 days, taking out rooted seedlings, cleaning culture medium at roots, transplanting into mixed matrix containing peat soil, perlite and vermiculite (3: 1: 1), and culturing in a greenhouse at 20-22 deg.C and relative humidity of above 80%.
After 30 days, the seedlings are transferred to an environment with the relative humidity of less than 80% for hardening, the water and soil are kept in a semi-dry state, and after 90 days, the seedlings have 3-5 leaves and the plant height reaches 10-15 cm.
The picture of the tissue culture seedling after 1 month of transplantation is shown in fig. 5, and the seedling grows vigorously.
After 3 months of transplanting, each plant had about 1-2 leaves, as shown in FIG. 6.
After 6 months of transplanting, each plant had about 2-4 leaves, as shown in FIG. 7.
After 2 years of transplanting, each plant has about 5-7 leaves, as shown in FIGS. 8-9. The tissue culture plants of 2 years are transplanted, the growth state of the roots is shown in figure 10, and the roots grow vigorously.
After 3 years of transplanting, the plant is about 30-50cm high and can flower and bear fruits, as shown in figure 11. FIG. 12 is a side view of a plant with roots exposed, where vigorous growth of the roots can be observed.
FIG. 13 shows the flowering condition of the plants transplanted for 3 years in the flowering stage, which shows that the female parent genetic traits of the excellent variety of peony are well maintained.
FIG. 14 shows the fruiting of plants transplanted for three years, the fruiting characteristics are the same as those of the female parent.
Example 2 tissue culture propagation of peony 2
The tissue culture propagation of the peony comprises the following steps:
(a) establishment of sterile System
Obtaining branches with sprout tillering buds and dormant buds on the current-year peony plants, rinsing with 1% of detergent water, washing with running water for 25 minutes, disinfecting with 75% of alcohol in an ultra-clean workbench for 35 seconds, and washing with sterile water for 3 times; the method comprises sterilizing for 2 times, treating with 0.5% sodium hypochlorite solution for 4 min, washing with sterile water for 2 times, soaking in 0.5% sodium hypochlorite solution for 4-6 min, and washing with sterile water for 3 times.
(b) Primary culture
Inoculating the dormant bud and the sprout bud with scales removed to a start culture medium, performing dark culture for 3 days, and then transferring to normal illumination culture.
The start-up medium contains: 1/2MS +450 mg/l calcium chloride +1.0 mg/l 6-benzyladenine (6-BA) +0.5 mg/l naphthylacetic acid (NAA) +0.8 mg/l gibberellin (GA3) +300 mg/l PVPP +0.8 mg/l silver nitrate.
And (3) carrying out primary culture for 15-20 days, and germinating a series of adventitious buds on the explants.
(c) Propagation and multiplication culture
After primary culture for 15-20 days, cutting 1.5-2 cm of adventitious bud, and inoculating to propagation medium.
The propagation and proliferation culture medium contains: MS +450 mg/l calcium chloride +1.0 mg/l 6-benzyladenine (6-BA) +0.20 mg/l indole-3-butyric acid (IBA) +6 mg/l hymexazol +0.8 mg/l gibberellin +400 mg/l PVPP +50 mg/l ascorbic acid (Vc).
And carrying out propagation and proliferation culture for 30-35 days, further inducing multiple shoots from the single shoot, wherein the number of the multiple shoots generated by one single shoot is about 4-6.
A part of the plants obtained by propagation and proliferation culture are shown in FIG. 2.
(d) Strong seedling culture
And transferring the cluster buds induced from the propagation culture medium into a strong seedling culture medium for culture.
The strong seedling culture medium contains: MS +450 mg/l calcium chloride +1.0 mg/l 6-benzyladenine (6-BA) +0.2 mg/l naphthylacetic acid (NAA) +55 mg/l ascorbic acid (Vc).
Culturing in strong seedling culture medium for 20-25 days to obtain strong bud with height of 2-3.5 cm.
(e) Rooting culture
Cutting the peony clump buds with high growth vigor and the height of about 2.5-3 cm into single buds, selecting robust single buds with more than 3 leaves, and inoculating the strong single buds into a rooting culture medium for culture. The cells were kept in the dark for 5 days before being transferred to light culture.
The rooting culture medium comprises: 1/2MS +450 mg/l calcium chloride +2 mg/l indole-3-butyric acid (IBA) +0.8 mg/l naphthylacetic acid (NAA) +1.5 g/l Activated Carbon (AC).
Rooting culture for about 15-20 days, wherein more than 90% of seedlings start to root, and 4-6 cm of roots grow in about 30-40 days, and each plant has 7-10 roots on average.
After rooting, rooted shoots are harvested from the culture vessel and are shown in FIG. 4 in a side view.
(f) Transplanting domestication
Selecting strong rooted seedlings with roots of about 4-7 cm and the strong rooted seedlings with strong roots for about 30-40 days, taking out the strong rooted seedlings from a culture room, hardening the seedlings in a greenhouse under scattered light at 20-22 ℃, and gradually removing bottle caps.
Transplanting after 7-10 days, taking out rooted seedlings, cleaning culture medium at roots, transplanting into mixed matrix containing peat soil, perlite and vermiculite (3: 1: 1), culturing in a greenhouse at 20-22 deg.C and relative humidity of above 80%.
After 30 days, the seedlings are transferred to an environment with the relative humidity of less than 80% for hardening, the water and soil are kept in a semi-dry state, and after 90 days, the seedlings have 3-5 leaves and the plant height reaches 10-15 cm.
Half a year later, 1-2 leaves, one year later, 2-4 leaves, two years later, 4-6 leaves, and three years later, 30-50cm high, and can blossom and bear fruit.
By adopting the method, the propagation coefficient is 4-6, a large number of robust tissue culture plants can be obtained in a short period, the root system is developed, the growth vigor is good, and the transplanting survival rate exceeds 90 percent.
The method can well keep the female parent genetic traits of excellent peony varieties by observing the overall morphology and the flower shape of the peony cultivated for three years.
Example 3 growth analysis in the propagation and proliferation culture stage
In this example, substantially the same culture conditions as in example 1 were used, but the difference was that: in the stage of propagation and propagation culture, the propagation and propagation culture medium is adjusted to a certain extent, and the effect of propagation and propagation culture is analyzed.
The results are shown in Table 5.
TABLE 5
Therefore, in the culture process, the application of a proper culture reagent is beneficial to promoting the growth of buds and improving the tissue culture efficiency.
Example 4 growth analysis in rooting culture
In this example, substantially the same culture conditions as in example 2 were used, but the difference was that: in the rooting culture stage, the rooting culture medium is regulated to some extent, and the rooting culture effect is analyzed.
The results are shown in Table 6.
TABLE 6
Therefore, during the culture process, the formula of the culture medium is adjusted to be beneficial to promoting the growth of roots.
Example 5 growth analysis in Primary culture
In this example, substantially the same culture conditions as in example 1 were used, but the difference was that: in the primary culture stage, the start medium is adjusted to some extent, and the effect of the culture in this stage is analyzed.
The results are shown in Table 7.
TABLE 7
Therefore, in the culture process, the application of a proper culture reagent is beneficial to promoting the growth of buds and improving the tissue culture efficiency.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (15)

1. A method for inducing peony to root, which is characterized by comprising the following steps:
(1) obtaining a stem section with buds of a peony explant, starting culture medium culture, and inducing to obtain adventitious buds; the start-up medium comprises: 1/2MS culture medium; 6-benzyladenine 0.5-1.0 mg/l; 0.20-0.50 mg/L of naphthylacetic acid; gibberellin 0.6-1.0 mg/L; PVPP 200-; 0.5-1.0 mg/L of silver nitrate; 400-600 mg/l calcium chloride;
(2) inoculating the adventitious bud induced in the step (1) into a propagation culture medium for culture, and inducing to generate cluster buds; the propagation culture medium comprises: MS culture medium; 6-benzyladenine 0.50-1.50 mg/l; indole-3 butyric acid 0.10-0.30 mg/l; hymexazol 5.0-7.0 mg/L; gibberellin 0.6-1.0 mg/L; PVPP 200-; ascorbic acid 40-60 mg/l; 400-600 mg/l calcium chloride;
(3) inoculating the cluster buds obtained in the step (2) into a strong seedling culture medium to obtain strong seedling; wherein, the strong seedling culture medium comprises: MS culture medium; 6-benzyladenine 0.50-1.50 mg/l; 0.10-0.30 mg/L of naphthylacetic acid; ascorbic acid 40-60 mg/l; 400-600 mg/l calcium chloride;
(4) cutting single buds from the bud cluster in the step (3), and culturing by using a rooting culture medium to obtain rooted test-tube plantlets; wherein, the rooting culture medium comprises: 1/2MS culture medium; indole-3 butyric acid 1.0-3.0 mg/l; 0.5-2.0 mg/L of naphthylacetic acid; 1.0-2.0 g/L of active carbon; 400-600 mg/l calcium chloride.
2. A transplanting domestication method of peony tissue culture seedlings is characterized by comprising the following steps:
(a) inducing by the method of claim 1 to obtain rooted peony test-tube plantlets;
(b) transplanting the rooted test-tube plantlet in the step (a) into a seedling culture substrate for seedling hardening;
(c) transplanting the acclimated seedlings of the step (b) to a natural environment.
3. The method according to claim 1 or 2, wherein in the step (1), the culture is performed in a dark culture for 3 to 4 days and then in a light culture, when the medium is started to be cultured; or
The time for starting the culture of the culture medium is 15-20 days.
4. The method of claim 1 or 2, wherein in step (2), the propagation medium is cultured for 25 to 35 days.
5. The method according to claim 1 or 2, wherein in the step (3), the period of culturing in the strong seedling medium is 20 to 30 days.
6. The method according to claim 1 or 2, wherein in the step (4), the rooting medium is cultured in a light culture for 30 to 40 days after 4 to 6 days in the dark.
7. The method of claim 2, wherein in step (b), the growth substrate comprises peat soil, perlite, vermiculite, or combinations thereof.
8. The method of claim 2, wherein in step (b), the subculture is carried out before seedling hardening at a temperature of 20-22 ℃ and a relative humidity of 80-90%.
9. The method of claim 2, wherein in step (b), the relative humidity during acclimatization is 80% or less.
10. The method of claim 1, wherein the medium further comprises sucrose and agar.
11. The method of claim 1 or 2, further comprising, prior to step (1), sterilizing the peony explants.
12. The method of claim 1 or 2, wherein 2-3 times the disinfection with sodium hypochlorite solution is performed.
13. The method of claim 12, wherein the explant is first rinsed with a detergent solution, then sterilized with an alcohol solution, and then sterilized with 0.5% sodium hypochlorite 2-3 times.
14. A kit for culturing peony, comprising: starting a culture medium, a propagation and proliferation culture medium, a strong seedling culture medium and a rooting culture medium; wherein the content of the first and second substances,
the start-up medium comprises: 1/2MS culture medium; 6-benzyladenine 0.5-1.0 mg/l; 0.20-0.50 mg/L of naphthylacetic acid; gibberellin 0.6-1.0 mg/L; PVPP 200-; 0.5-1.0 mg/L of silver nitrate; 400-600 mg/l calcium chloride;
the propagation and proliferation culture medium comprises: MS culture medium; 6-benzyladenine 0.50-1.50 mg/l; indole-3 butyric acid 0.10-0.30 mg/l; hymexazol 5.0-7.0 mg/L; gibberellin 0.6-1.0 mg/L; PVPP 200-; ascorbic acid 40-60 mg/l; 400-600 mg/l calcium chloride;
the strong seedling culture medium comprises: MS culture medium; 6-benzyladenine 0.50-1.50 mg/l; 0.10-0.30 mg/L of naphthylacetic acid; ascorbic acid 40-60 mg/l; 400-600 mg/l calcium chloride;
the rooting medium comprises: 1/2MS culture medium; indole-3 butyric acid 1.0-3.0 mg/l; 0.5-2.0 mg/L of naphthylacetic acid; 1.0-2.0 g/L of active carbon; 400-600 mg/l calcium chloride.
15. The kit of claim 14, further comprising a culture medium selected from the group consisting of: sucrose, agar.
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