CN116250480B - Sterile rapid propagation method of trollius chinensis and planting method of trollius chinensis - Google Patents
Sterile rapid propagation method of trollius chinensis and planting method of trollius chinensis Download PDFInfo
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 41
- 239000005708 Sodium hypochlorite Substances 0.000 claims abstract description 35
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
- A01C1/08—Immunising seed
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/40—Afforestation or reforestation
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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Environmental Sciences (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Soil Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The application discloses a method for sterile rapid propagation of trollius chinensis and a planting method of trollius chinensis, and relates to the technical field of trollius chinensis cultivation. The sterile rapid propagation method comprises the steps of disinfecting cleaned trollius chinensis seeds by adopting alcohol and sodium hypochlorite together; then inoculating and germinating to obtain trollius chinensis seedlings; wherein, the germination medium is obtained by adding cephalosporin and mixed solution into a basic medium, and then adding active carbon and vitamin C; culturing trollius chinensis bunge seedling, hardening and transplanting. The treatment can effectively solve the problems of poor disinfection effect, serious bacteria infection and browning of the trollius chinensis and is beneficial to improving the seedling rate and proliferation rate of trollius chinensis tissue culture. Meanwhile, the method provided by the application starts germination after 5-6 days, the sterile germination rate of the seeds is 80.67%, the propagation period is 60 days, the propagation coefficient is 4.34, the transplanting survival rate is more than 90%, and the propagation quantity and growth rate of trollius chinensis are greatly improved.
Description
Technical Field
The invention relates to the technical field of trollius chinensis cultivation, in particular to a trollius chinensis sterile rapid propagation method and a trollius chinensis planting method.
Background
Flos Trollii is a perennial plant of Ranunculaceae, and its flower has high medicinal value, and can be used as medicine or tea. In recent years, with the improvement of people on the understanding of medicinal value and ornamental value of trollius chinensis bunge, the picking quantity of trollius chinensis bunge is increased year by year, and the wild resources of trollius chinensis bunge are seriously destroyed. Therefore, the method has great significance in introduction and domestication and artificial cultivation of the wild trollius chinensis.
The sexual reproduction of trollius chinensis has the common problems of low germination rate, poor survival rate of seedlings and the like. The tissue culture technology is an effective way for plant resource conservation and development, can save plant germplasm resources in vitro through tissues and organs, and can reduce the demand for wild resources by providing a large number of seedlings for industrial seedling culture.
Most of the existing trollius chinensis tissue culture technologies adopt 0.1% mercuric chloride for sterilizing the explant, but the mercuric chloride belongs to a highly toxic medicament, residues are not easy to remove, the activity of the explant is killed, and serious damage is caused to the environment. When the trollius chinensis callus induction way is adopted for tissue culture, the yield is low, the browning is serious, the callus is difficult to redifferentiate, the regeneration system is not stable enough, and the complete plant is difficult to induce. When tissue culture is carried out by adopting a trollius chinensis sterile seed rapid propagation way, the sterilization effect of a sterilizing agent on seeds is mostly considered, and the influence of the sterilizing agent on the activity of explants is ignored, so that the seed seedling rate is low. While disregarding the browning that accompanies germination and proliferation culture. In addition, the tissue culture of trollius chinensis is only carried out in a laboratory stage at present, and the tissue culture system has the phenomena of high cost and no optimization.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a sterile rapid propagation method of trollius chinensis, which can reduce the pollution rate in the seed germination process, can also relieve the problem of seedling browning, and achieves good cultivation effect at lower cost.
The invention aims to provide a planting method of trollius chinensis bunge.
The invention is realized in the following way:
in a first aspect, the present invention provides a method for asepsis rapid propagation of trollius chinensis, comprising:
Sterilizing the cleaned trollius chinensis seeds with 75% alcohol for 1-2min, and then sterilizing with 4-6% sodium hypochlorite for 20-30min to obtain sterile seeds;
Inoculating the sterile seeds into a germination culture medium for germination to obtain trollius chinensis germination seedlings; wherein, the germination medium is obtained by adding 20-30mg/L of cephalosporin, 20-40mg/L of mixed solution of 65-75% mancozeb and 70-80% chlorothalonil 1:1 into a basic medium, and then adding 40-60mg/L of active carbon and 80-120mg/L of vitamin C;
And artificially culturing the trollius chinensis bunge sprouting seedlings to obtain tissue culture seedlings, and hardening and transplanting the tissue culture seedlings.
In an alternative embodiment, the basal medium is obtained by replacing sucrose in an MS medium with white sugar 23-27mg/L and reducing the agar dosage to 5-6 g/L.
In an alternative embodiment, 1.0-2.5 mg/L6-BA and 0.1-0.5mg/L NAA are also added to the basal medium.
In an alternative embodiment, artificially culturing the trollius chinensis bunge sprouting seedling to obtain a tissue culture seedling comprises:
Transplanting the trollius chinensis bunge sprouting seedlings to a sterilized sprouting culture medium for continuous artificial sprouting culture to obtain trollius chinensis bunge aseptic seedlings, wherein the artificial sprouting culture is carried out for seven days in a dark culture mode, and then 12 h/12 h of light is carried out, 1300-1700Lux of light intensity is carried out, and the sprouting culture time is 28-30d;
taking the stem segments of the trollius chinensis aseptic seedlings as explants, and inserting the explants into proliferation and subculture media for proliferation and subculture to obtain clustered bud stem segments; and inoculating the cluster bud stem sections into a rooting culture medium for culture to obtain the tissue culture seedlings.
In an alternative embodiment, the proliferation and subculture medium is obtained by adding 1.5-2.5 mg/L6-BA and 0.3-0.4mg/L NAA to MS medium, and further adding 40-60mg/L activated carbon and 80-120mg/L vitamin C, and the proliferation and subculture time is 58-62d.
In an alternative embodiment, the rooting medium is obtained by adding 0.1-0.5mg/L NAA to 1/2MS medium, and then adding 40-60mg/L activated carbon and 80-120mg/L vitamin C, wherein the rooting culture time is 28-32d.
In an alternative embodiment, hardening and transplanting the tissue culture seedling comprises: placing the tissue culture seedlings in the tissue culture bottle in advance for 5 days in a greenhouse for hardening seedlings, wherein the matrix is a mixture of turfy soil and perlite, and the turfy soil in the mixture is as follows: the volume ratio of the perlite is 3:1, sterilizing at high temperature in a sterilizing pot, regulating the pH value to 5.8, taking out the tissue culture seedlings from the tissue culture bottles, cleaning the culture medium on the roots with water, soaking the culture medium in 1% carbendazim for 10min, and then planting the culture medium in a plug tray.
In an alternative embodiment, washing the trollius chinensis seeds comprises: wrapping the trollius chinensis seeds in sterilized gauze, soaking and cleaning in a detergent for 15-25min, and then washing with clear water for 25-35min.
In a second aspect, the present invention provides a planting method of trollius chinensis, which includes the method for sterile rapid propagation of trollius chinensis according to any one of the foregoing embodiments.
The invention has the following beneficial effects:
The sterile rapid propagation method of trollius chinensis provided by the application adopts 75% alcohol and 2-10% sodium hypochlorite to disinfect trollius chinensis seeds, has good disinfection effect and does not have adverse effect on the activity of an explant and the environment. Furthermore, 20-30mg/L of cephalosporin and 20-40mg/L of mixed solution of 65-75% mancozeb and 70-80% chlorothalonil 1:1 are also added into the basic culture medium for disinfection, so that the pollution rate of seed germination can be further reduced, and the survival rate of seedlings is not influenced. And the phenomenon of secondary bacteria infection caused by endophyte generated by the trollius chinensis per se or incomplete sterilization can not occur in the subsequent experiments. The addition of the activated carbon and the vitamin C can play a role in synergy, not only can effectively improve browning, but also can avoid influencing the growth and differentiation of seedlings. The method can effectively solve the problems of poor disinfection effect, bacteria staining phenomenon and serious browning imagination of the trollius chinensis bunge, and is beneficial to improving the seedling rate of trollius chinensis bunge tissue culture. The application adopts the method of sterile germination and rapid propagation of trollius chinensis seeds to obtain offspring of the same seedling single plant line, has easy seedling formation, simplified propagation steps and high effective propagation rate, and has great significance for preserving and expanding trollius chinensis population. The germination rate of the trollius chinensis seeds bred by the sterile germination and rapid propagation method is 80.67% after 30 days, the propagation coefficient is 4.34 within 60 days, the transplanting survival rate is more than 90%, the propagation coefficient of trollius chinensis is greatly improved, and a very effective propagation method is provided for the introduction and domestication of the species, the preservation, the garden gardening and the utilization of scientific research value of the species.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
Fig. 1 is a schematic diagram of germination conditions of trollius chinensis bunge provided in embodiment 1 of the present application;
Fig. 2 is a schematic diagram of the artificial cultivation of trollius chinensis bunge provided in embodiment 1 of the present application to form sterile seedlings;
fig. 3 is a schematic diagram of tissue culture propagation conditions of trollius chinensis bunge provided in embodiment 1 of the present application;
Fig. 4 is a schematic diagram of a trollius chinensis tissue culture rooting condition provided in example 1 of the present application.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The invention provides a method for sterile rapid propagation of trollius chinensis, which comprises the following steps:
S1, obtaining sterile seeds.
The trollius chinensis seeds are wrapped in the sterilized gauze, so that the leakage phenomenon of the seeds during oscillation can be effectively reduced. Soaking and cleaning in the detergent for 15-25min, and then washing with clear water for 25-35min.
And (3) transferring the cleaned trollius chinensis seeds to an ultra-clean workbench, sterilizing for 1-2min by adopting 75% alcohol, and then sterilizing for 20-30min by adopting 2-10% sodium hypochlorite to obtain sterile seeds. It has been found that the existing trollius chinensis seed disinfection can reduce the pollution rate but also affect the survival rate of seeds by adopting 0.1% mercuric chloride for explant disinfection. In the application, the sterilization is sequentially carried out by adopting a mode of combining 75% of alcohol and 2-10% of sodium hypochlorite, the effect is optimal, and the activity and the environment of the explant are not adversely affected. In this example, 75% alcohol is not primarily intended for disinfection, but rather to facilitate wetting of the plant surface; sodium hypochlorite is the primary disinfectant. The 75% alcohol is to allow sodium hypochlorite to act better on the seed surface. When the alcohol concentration is too high or too low, the disinfection and sterilization effects are poor, and the concentration of sodium hypochlorite is too high or too low, so that disinfection and sterilization are not facilitated.
S2, inoculating the sterile seeds into a germination culture medium for germination to obtain trollius chinensis germination seedlings.
In the application, the basal medium is obtained by taking an MS medium as a base, replacing sucrose in the MS medium with white sugar 23-27mg/L, and reducing the agar dosage to 5-6 g/L. The specific components of the MS medium are known, and the application is not specifically described.
The germination medium is prepared by adding 20-30mg/L of cephalosporin and 20-40mg/L of mixed solution of 65-75% mancozeb and 70-80% chlorothalonil in a ratio of 1:1 into a basic medium, and then adding 40-60mg/L of active carbon and 80-120mg/L of vitamin C into the basic medium after disinfection.
Normally, the culture medium is inoculated after being directly subjected to conventional sterilization (such as high-temperature sterilization), however, in the application, the pollution rate of seed germination can be further reduced by adding 20-30mg/L of cephalosporin and 20-40mg/L of a mixed solution of 65-75% mancozeb and 70-80% chlorothalonil 1:1 into the basic culture medium on the basis of conventional sterilization, and the survival rate of seedlings can not be influenced. And the phenomenon of secondary bacteria infection caused by endophyte generated by the trollius chinensis per se or incomplete sterilization can not occur in the subsequent experiments. The existing trollius chinensis tissue culture process can be accompanied with the phenomenon of bacteria infection.
The trollius chinensis contains polyphenol compounds, and wounds when buds are cut can activate polyphenol oxidase in tissues, oxidize polyphenol substances into tan quinone substances, brown the cut of an explant, permeate the cut of the explant into a culture medium, brown the culture medium, and seriously affect the growth and differentiation of the culture, and even cause death of the culture. According to the application, the addition of the activated carbon and the vitamin C can improve the problem that partial seedlings are brown in the culture process. Wherein, the active carbon can absorb brown quinone substances with inhibition effect on growth, and the vitamin C can be used as an oxidant to effectively prevent browning.
It should be understood that although proliferation and subculture cluster bud induction are more prone to browning, all experimental procedures are likely to be brown, and thus, the present application also adds activated carbon and vitamin C to all media after screening the optimal anti-browning regimen. Furthermore, in the application, 1.5-2.5 mg/L6-BA and 0.1-0.2mg/L NAA are also added into the basic culture medium, and the growth hormone is added in the germination process of the sterile seeds, so that the germination rate can be promoted, and the germination rate is up to 80.67%.
S3, artificially culturing the trollius chinensis bunge sprouting seedlings to obtain tissue culture seedlings.
(1) Transplanting trollius chinensis bunge sprouting seedlings into a sterilized optimal sprouting culture medium, inoculating 6-8 trollius chinensis bunge sprouting seedlings in each bottle, and performing artificial culture in an artificial culture room with the temperature of 25+/-2 ℃ and the humidity of 70% and the photoperiod of 12h illumination/12 h darkness to obtain trollius chinensis bunge aseptic seedlings;
(2) Cutting the leaves into stem segments containing axillary bud nodes on sterile filter paper by using a scalpel, taking the stem segments of trollius chinensis aseptic seedlings as explants, and inserting the explants into proliferation and subculture media under the culture conditions: culturing in an artificial culture room with the temperature of 25+/-2 ℃ and the humidity of 70% and the photoperiod of 12h illumination/12 h darkness to obtain clustered bud stem segments; wherein the proliferation and subculture medium is obtained by adding 1.5-2.5 mg/L6-BA and 0.3-0.4mg/L NAA, and then adding 40-60mg/L active carbon and 80-120mg/L vitamin C into MS culture medium.
(3) Inoculating the cluster bud stem sections into a rooting culture medium for culture to obtain tissue culture seedlings, wherein the rooting culture medium is obtained by adding 0.4-0.6mg/L NAA, 40-60mg/L active carbon and 80-120mg/L vitamin C into a 1/2MS culture medium.
S4, hardening and transplanting the tissue culture seedlings.
The seedling hardening is carried out before the trollius chinensis tissue culture seedling is transplanted, so that the resistance of the tissue culture seedling to the adverse environment can be improved, and the survival rate of the transplanted seedling can be improved. After the trollius chinensis tissue culture seedling grows root for 30d, the tissue culture seedling is transferred into a greenhouse for natural light irradiation (strong light is prevented from being directly irradiated), the indoor temperature is close to the temperature of a culture room (23 ℃ -25 ℃), and the seedling is allowed to be slowly adapted to the external environment conditions, so that the adaptability of the trollius chinensis tissue culture seedling to the external environment is improved. And (3) half opening the bottle cap after 2d, adding a little sterile water to cover the surface layer of the culture medium, standing for two days, completely opening the bottle cap after 3d, and standing for 3d-5d to obtain the transplanting. And then taking out the trollius chinensis tissue culture seedlings from the tissue culture bottle, and cleaning the culture medium on the roots with water to prevent diseases such as bacteria. The root system is not hurt during cleaning, then 1% carbendazim is used for soaking for 10min, and then the plant is planted in the plug. A small hole is pricked in the middle of the hole of the cave dish, the root system is inserted in, the substrate turfy soil and perlite are mixed with the substrate, the ratio is 3:1 (high temperature sterilization is carried out in a sterilizing pot), the root system is covered, and water is poured thoroughly. And 1 seedling is planted in 1 hole during transplanting. After the transplanting is completed, the seedlings are placed in a greenhouse for shading culture for a few days, and then normal culture is carried out. The survival rate of the seedling hardening and transplanting reaches more than 90 percent.
In a second aspect, the present invention provides a planting method of trollius chinensis, which comprises the sterile rapid propagation method of trollius chinensis according to any one of the previous embodiments.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
The embodiment provides a method for sterile rapid propagation of trollius chinensis, which comprises the following steps:
S1, obtaining sterile seeds.
Wrapping flos Trollii seed in sterilized gauze, soaking and cleaning in detergent for 20min, and then washing with clear water for 30min. And (3) transferring the cleaned trollius chinensis seeds to an ultra-clean workbench, sterilizing for 1min by adopting 75% alcohol, and sterilizing for 30min by adopting 5% sodium hypochlorite to obtain sterile seeds.
S2, inoculating the sterile seeds into a germination culture medium for germination to obtain trollius chinensis bunge germinated seedlings (refer to fig. 1).
Firstly, taking an MS culture medium as a base, replacing sucrose in the MS culture medium with 25mg/L of white sugar, reducing the agar dosage to 6g/L to obtain a basic culture medium, sterilizing the basic culture medium at high temperature, then adding 25mg/L of cephalosporin, 30mg/L of 70% mancozeb and 75% chlorothalonil 1:1 mixed solution into the basic culture medium for sterilization, and after sterilization, adding 50mg/L of active carbon and 100mg/L of vitamin C into the basic culture medium, and then adding 2mg/L of 6-BA and 0.1mg/L of NAA to obtain a germination culture medium.
S3, artificially culturing the trollius chinensis bunge sprouting seedlings to obtain tissue culture seedlings.
(1) Transplanting the trollius chinensis bunge sprouting seedlings into a sterilized optimal sprouting culture medium, inoculating 6-8 trollius chinensis bunge sprouting seedlings in each bottle, and performing artificial culture in an artificial culture room with the temperature of 25+/-2 ℃ and the humidity of 70% and the photoperiod of 12h illumination/12 h darkness to obtain trollius chinensis bunge aseptic seedlings (refer to figure 2).
(2) Cutting the leaves into stem segments containing axillary bud nodes on sterile filter paper by using a scalpel, taking the stem segments of trollius chinensis aseptic seedlings as explants, and inserting the explants into proliferation and subculture media under the culture conditions: culturing in an artificial culture room with temperature of 25+ -2deg.C, humidity of 70% and photoperiod of 12h illumination/12 h darkness to obtain clustered bud stem segments (refer to figure 3); wherein, the proliferation and secondary culture medium is obtained by adding 2 mg/L6-BA and 0.3mg/L NAA, and further adding 50mg/L active carbon and 100mg/L vitamin C into MS culture medium.
(3) The clustered shoots and stems were inoculated into rooting medium (see FIG. 4) obtained by adding 0.5mg/L NAA to 1/2MS medium, followed by 50mg/L activated carbon and 100mg/L vitamin C.
S4, hardening and transplanting the tissue culture seedlings.
The seedling hardening is carried out before the trollius chinensis tissue culture seedling is transplanted, so that the resistance of the tissue culture seedling to the adverse environment can be improved, and the survival rate of the transplanted seedling can be improved. After the trollius chinensis tissue culture seedling grows root for 30d, the tissue culture seedling is transferred into a greenhouse for natural light irradiation (strong light is prevented from being directly irradiated), the indoor temperature is close to the temperature of a culture room (23 ℃ -25 ℃), and the seedling is allowed to be slowly adapted to the external environment conditions, so that the adaptability of the trollius chinensis tissue culture seedling to the external environment is improved. And (3) half opening the bottle cap after 2d, adding a little sterile water to cover the surface layer of the culture medium, standing for two days, completely opening the bottle cap after 3d, and standing for 3d-5d to obtain the transplanting. And then taking out the trollius chinensis tissue culture seedlings from the tissue culture bottle, and cleaning the culture medium on the roots with water to prevent diseases such as bacteria. The root system is not hurt during cleaning, then 1% carbendazim is used for soaking for 10min, and then the plant is planted in the plug. A small hole is pricked in the middle of the hole of the plug tray, the root system is introduced, and the matrix turfy soil and perlite are mixed with the matrix, the proportion is 3:1 (high temperature sterilization in a sterilizing pot) covering the root system, and watering thoroughly. And 1 seedling is planted in 1 hole during transplanting. After the transplanting is completed, the seedlings are placed in a greenhouse for shading culture for a few days, and then normal culture is carried out. The survival rate of the seedling hardening and transplanting reaches more than 90 percent.
Example 2
The embodiment provides a method for sterile rapid propagation of trollius chinensis, which comprises the following steps:
S1, obtaining sterile seeds.
Wrapping flos Trollii seed in sterilized gauze, soaking and cleaning in detergent for 15min, and then washing with clear water for 35min. And (3) transferring the cleaned trollius chinensis seeds to an ultra-clean workbench, sterilizing for 2min by adopting 75% alcohol, and sterilizing for 20min by adopting 5% sodium hypochlorite to obtain sterile seeds.
S2, inoculating the sterile seeds into a germination culture medium for germination to obtain trollius chinensis seedlings.
Firstly, taking an MS culture medium as a base, replacing sucrose in the MS culture medium with 23mg/L white sugar, reducing the agar dosage to 5g/L to obtain a basic culture medium, sterilizing the basic culture medium at high temperature, then adding 25mg/L cephalosporin, 20mg/L75% mancozeb and 80% chlorothalonil 1:1 mixed solution into the basic culture medium for sterilization, and after sterilization, adding 40mg/L active carbon and 120mg/L vitamin C into the basic culture medium, and then adding 1.5 mg/L6-BA and 0.1mg/L NAA to obtain a germination culture medium.
S3, artificially culturing the trollius chinensis bunge sprouting seedlings to obtain tissue culture seedlings.
(1) Transplanting the trollius chinensis bunge sprouting seedlings into a sterilized optimal sprouting culture medium, inoculating 6-8 trollius chinensis bunge sprouting seedlings in each bottle, and performing artificial culture in an artificial culture room with the temperature of 25+/-2 ℃ and the humidity of 70% and the photoperiod of 12h illumination/12 h darkness to obtain the trollius chinensis bunge aseptic seedlings.
(2) Cutting the leaves into stem segments containing axillary bud nodes on sterile filter paper by using a scalpel, taking the stem segments of trollius chinensis aseptic seedlings as explants, and inserting the explants into proliferation and subculture media under the culture conditions: culturing in an artificial culture room with the temperature of 25+/-2 ℃ and the humidity of 70% and the photoperiod of 12h illumination/12 h darkness to obtain clustered bud stem segments; wherein, the proliferation and secondary culture medium is obtained by adding 1.5 mg/L6-BA and 0.3mg/L NAA, and then adding 40mg/L active carbon and 120mg/L vitamin C into MS culture medium.
(3) Inoculating the cluster bud stem segments into a rooting culture medium for culture to obtain tissue culture seedlings, wherein the rooting culture medium is obtained by adding 0.4mg/L NAA, 40mg/L active carbon and 120mg/L vitamin C into a 1/2MS culture medium.
S4, hardening and transplanting the tissue culture seedlings by adopting the same method as in the embodiment 1.
Example 3
The embodiment provides a method for sterile rapid propagation of trollius chinensis, which comprises the following steps:
S1, obtaining sterile seeds.
Wrapping flos Trollii seed in sterilized gauze, soaking and cleaning in detergent for 25min, and then washing with clear water for 25min. And (3) transferring the cleaned trollius chinensis seeds to an ultra-clean workbench, sterilizing for 1min by using 75% alcohol, and sterilizing for 25min by using 6% sodium hypochlorite to obtain sterile seeds.
S2, inoculating the sterile seeds into a germination culture medium for germination to obtain trollius chinensis germination seedlings.
Firstly, taking an MS culture medium as a base, replacing sucrose in the MS culture medium with 27mg/L white sugar, reducing the agar dosage to 6g/L to obtain a basic culture medium, sterilizing the basic culture medium at high temperature, then adding 25mg/L cephalosporin, 40 mg/L65% mancozeb and 70% chlorothalonil 1:1 mixed solution into the basic culture medium for sterilization, and after sterilization, adding 60mg/L active carbon and 80mg/L vitamin C into the basic culture medium, and then adding 2.5 mg/L6-BA and 0.2mg/L NAA to obtain a germination culture medium.
S3, artificially culturing the trollius chinensis bunge sprouting seedlings to obtain tissue culture seedlings.
(1) Transplanting the trollius chinensis bunge sprouting seedlings into a sterilized optimal sprouting culture medium, inoculating 6-8 trollius chinensis bunge seedlings in each bottle, and performing artificial culture in an artificial culture room with the temperature of 25+/-2 ℃ and the humidity of 70% and the photoperiod of 12h illumination/12 h darkness to obtain the trollius chinensis bunge aseptic seedlings.
(2) Cutting the leaves into stem segments containing axillary bud nodes on sterile filter paper by using a scalpel, taking the stem segments of trollius chinensis aseptic seedlings as explants, and inserting the explants into proliferation and subculture media under the culture conditions: culturing in an artificial culture room with the temperature of 25+/-2 ℃ and the humidity of 70% and the photoperiod of 12h illumination/12 h darkness to obtain clustered bud stem segments; wherein, the cluster bud culture medium is obtained by adding 2.5 mg/L6-BA and 0.4mg/L NAA, and then adding 60mg/L active carbon and 80mg/L vitamin C into MS culture medium.
(3) Inoculating the cluster bud stem segments into a rooting culture medium for culture to obtain tissue culture seedlings, wherein the rooting culture medium is obtained by adding 0.6mg/L NAA, 60mg/L active carbon and 80mg/L vitamin C into a 1/2MS culture medium.
S4, hardening and transplanting the tissue culture seedlings by adopting the same method as in the embodiment 1.
Comparative example 1
In this comparative example, "sterilization with 75% alcohol for 1min and then with 5% sodium hypochlorite for 30min" in step S1 of example 1 was replaced with "sterilization with 0.1% mercuric chloride".
Since mercury is a hazardous material, the results of this comparative example were obtained by reference to the literature: the sterilization effect achieved by using 0.1% mercuric chloride for sterilization is better than that achieved by using 5% sodium hypochlorite for sterilization for 30min, but the mercuric chloride is extremely toxic and can pollute the body and the environment, and when the 0.1% mercuric chloride is used for sterilization of trollius chinensis seeds, the germination rate and the survival rate of trollius chinensis seeds are reduced although the bacterial infection rate is reduced, the 0.1% mercuric chloride is used for sterilization of trollius chinensis seeds for 20min, the bacterial infection rate of trollius chinensis is 6.05%, but the germination rate of trollius chinensis seeds is only 22.55%.
Comparative examples 2 to 4
In this comparative example, "disinfect with 75% alcohol for 1min and then with 5% sodium hypochlorite for 30min" of step S1 in example 1 was modified to "disinfect with 75% alcohol for 1min and then with 2% sodium hypochlorite for 10, 20 and 30min".
Wherein, the pollution rate of the trollius chinensis seeds obtained in the comparative example 2 is 62.22%, and the germination rate is 34.45%. The pollution rate of the trollius chinensis seeds obtained in the comparative example 3 is 53.89%, and the germination rate is 46.12%. The pollution rate of the trollius chinensis seeds obtained in the comparative example 4 is 47.78%, and the germination rate is 58.89%.
Comparative example 5
In this comparative example, "disinfect with 75% alcohol for 1min and then with 5% sodium hypochlorite for 30min" in step S1 of example 1 was modified to "disinfect with 75% alcohol for 1min and then with 5% sodium hypochlorite for 10min". The pollution rate of the obtained trollius chinensis seeds is 35.56%, and the germination rate is 38.89%.
Comparative examples 6 to 8
In this comparative example, "disinfect with 75% alcohol for 1min and then with 5% sodium hypochlorite for 30min" of step S1 in example 1 was modified to "disinfect with 75% alcohol for 1min and then with 10% sodium hypochlorite for 10, 20 and 30min".
Wherein, the pollution rate of the trollius chinensis seeds obtained in the comparative example 6 is 12.78%, and the germination rate is 38.89%. The pollution rate of the trollius chinensis seeds obtained in the comparative example 7 is 1.11%, and the germination rate is 28.89%. The pollution rate of the trollius chinensis seeds obtained in the comparative example 8 is 0%, a good sterilization effect is achieved, but the germination rate of the trollius chinensis seeds is greatly reduced, and the germination rate is 13.89%.
Comparative example 9
In this comparative example, "disinfect with 75% alcohol for 1min and then with 5% sodium hypochlorite for 30min" in step S1 of example 1 was modified to "disinfect with 5% sodium hypochlorite for 30min".
The pollution rate of the obtained trollius chinensis seeds is 24.67% and the germination rate is 60.67% after the trollius chinensis seeds are sterilized for 30min by using 5% sodium hypochlorite.
Comparative example 10
In the comparative example, the step S1 of "sterilizing by adding 25mg/L of cephalosporin and 30mg/L of a 1:1 mixture of 70% mancozeb and 75% chlorothalonil to a basal medium" in example 1 was omitted, and only high-temperature sterilization was used.
When the basal medium is sterilized at high temperature, the trollius chinensis tissue culture process can generate viscous liquid substances around the culture material and mildew at the parts of the culture base, so as to generate the bacterial contamination phenomena of villus hyphae, pathogenic spores and the like.
Comparative example 11
In this comparative example, the "sterilization by adding 25mg/L of cephalosporin to basal medium and 30mg/L of a 1:1 mixture of 70% mancozeb and 75% chlorothalonil" of step S1 in example 1 was modified to "sterilization by adding 25mg/L of cephalosporin to basal medium".
In the process of tissue culture of trollius chinensis bunge, the subsection of the culture basal part is mildewed, and the phenomenon of bacterial contamination such as villous hyphae, pathogenic spores and the like is generated.
Comparative example 12
In the comparative example, the "disinfection of a mixture of 25mg/L of cephalosporin and 30mg/L of 70% mancozeb and 75% chlorothalonil 1:1 added to a basal medium" of step S1 in example 1 was modified by "disinfection of a mixture of 30mg/L of 70% mancozeb and 75% chlorothalonil 1:1 added to a basal medium".
In the process of trollius chinensis tissue culture, turbid water stains appear on the culture medium, and sometimes, even a viscous liquid appears around the whole culture material.
Comparative example 13
In this comparative example, "Add 50mg/L activated carbon and 100mg/L vitamin C" of step S1 in example 1 was deleted.
The stem segments of the early lotus flowers are relatively heavy in browning, the cluster buds grow weakly, and the later lotus flowers are all brown.
Comparative example 14
In this comparative example, "50 mg/L activated carbon and 100mg/L vitamin C added" in step S1 of example 1 was replaced with "50 mg/L activated carbon added".
The trollius chinensis leaves turn yellow, the seedlings grow weaker, the browning rate reaches 65%, and the browning degree is heavier.
Comparative example 15
In this comparative example, "40-60 mg/L activated carbon and 80-120mg/L vitamin C added" in step S1 of example 1 was replaced with "100mg/L vitamin C".
The trollius chinensis bunge seedlings grow well, the browning rate is 30%, and the browning degree is moderate.
Comparative example 16
In this comparative example, "germination medium 2 mg/L6-BA and 0.1mg/L NAA" of step S1 in example 1 was replaced with "0.1mg/LTDZ".
The germination rate of the obtained trollius chinensis seeds is 44.67%, and the leaves of germinated seedlings are emerald green and the roots are thin and long.
Comparative example 17
In this comparative example, "germination medium 2 mg/L6-BA and 0.1mg/L NAA" of step S1 in example 1 was replaced with "0.1mg/LIBA".
The germination rate of the obtained trollius chinensis seeds is 30.67%, the germinated seedlings have no shrink leaves, and the leaves are light green.
Experimental example one: and (5) carrying out experiments on the pollution rate and germination rate of trollius chinensis seeds.
Contamination rate experiments were performed on the sterilization methods of examples 1-2 and comparative examples 1-9, and the experimental results are shown in Table 1.
TABLE 1 pollution Rate experiment results statistics table of trollius chinensis seeds
| Treatment of | Treatment method | Pollution rate (%) | Germination rate (%) |
| Example 1 | 75% Ethanol 1min,5% sodium hypochlorite 30min | 10.55±2.56f | 76.67±2.89a |
| Example 2 | 75% Ethanol 1min,5% sodium hypochlorite 20min | 20.00±3.34e | 57.23±0.96b |
| Comparative example 1 | 0.1% Mercuric chloride for 20min | 6.05 | 22.55 |
| Comparative example 2 | 75% Ethanol 1min,2% sodium hypochlorite 10min | 62.22±3.85a | 34.45±4.19de |
| Comparative example 3 | 75% Ethanol 1min,2% sodium hypochlorite 20min | 53.89±4.20b | 46.12±4.19c |
| Comparative example 4 | 75% Ethanol 1min,2% sodium hypochlorite 30min | 47.78±1.92c | 58.89±2.55b |
| Comparative example 5 | 75% Ethanol 1min,5% sodium hypochlorite 10min | 35.56±3.85d | 38.89±1.92d |
| Comparative example 6 | 75% Ethanol 1min,10% sodium hypochlorite 10min | 12.78±1.92f | 38.89±4.20d |
| Comparative example 7 | 75% Ethanol 1min,10% sodium hypochlorite 20min | 1.11±1.93g | 28.89±2.55e |
| Comparative example 8 | 75% Ethanol 1min,10% sodium hypochlorite 30min | 0g | 13.89±0.96f |
| Comparative example 9 | 5% Sodium hypochlorite sterilization for 30min | 24.67 | 60.67 |
It should be noted that, table 1 mainly performs the data processing of one-factor analysis of variance for the processing methods of ethanol+sodium hypochlorite of examples 1-2 and comparative examples 2-8, whereas the data of comparative examples 1 and comparative example 9 did not perform the data processing of one-factor analysis of variance, so only the corresponding contamination rate and germination rate are listed, and the basic judgment of all the data is not affected.
Further, the optimal germination medium was selected by varying the amounts of 6-BA and NAA added to the different germination media. The screening results are shown in Table 2.
TABLE 2 germination Rate experiment of trollius chinensis seeds
Based on the above screening, the amounts of 6-BA and NAA added to the germination medium in the present application are preferably 2 mg/L6-BA and 0.1mg/L NAA.
Experimental example two: and (3) a trollius chinensis tissue culture bacterial infection experiment.
As the trollius chinensis carries endophytes and the artificial and environmental pollution is caused by incomplete sterilization, the trollius chinensis can be infected during the tissue culture process, and common bacteria include bacteria in turbid water stains and mucus and fungi in villus, and various pathogenic spores are generated. The mixed solution of the antibiotic cephalosporin with good effect on resisting bacteria of the culture medium and the mancozeb and chlorothalonil with extremely strong contact fungicidal effect is added into the mixed solution, so that the risk of the tissue culture material being infected with bacteria can be remarkably reduced, and the growth and development of trollius chinensis are basically not influenced.
Experimental example three: and (5) performing seedling browning experiments in the culture process.
By varying the amounts of vitamin C and activated carbon added, optimum conditions for browning were obtained, operating conditions and results are shown in Table 3.
TABLE 3 screening of optimal conditions for browning
From the above table, it can be seen that the unexpected technical effect can be obtained by adopting the combination of activated carbon and vitamin C.
Experimental example four: proliferation rate experiments.
TABLE 4 proliferation rate experiment result statistics table
As can be seen from Table 4, the preferred proliferation and subculture medium of the present application comprises the following components: MS culture medium is added with 2 mg/L6-BA and 0.3-0.5mg/L NAA.
Experimental example five: rooting rate and transferring survival rate.
TABLE 5 trollius chinensis rooting rate experiment statistics table
| Auxin species and concentration | Rooting rate/% | Average root number/bar | Root characteristics |
| 0.1mg/LNAA | 60 | 4.5 | Root is thick and white adventitious root |
| 0.5mg/LNAA | 100 | 8.1 | The root is thick and strong, and has yellow fluff |
| 0.1mg/LIBA | 66.67 | 4.67 | The root is slender and has yellow fluff |
| 0.5mg/LIBA | 86.67 | 6.8 | The root is slender and has yellow fluff |
As can be seen from the above table, the effect obtained by adding the LNAA auxin in this example is significantly better than other auxins.
Experimental example six: trollius chinensis transplanting survival rate experiment
TABLE 6 transfer survival rate statistics table
| Matrix protocol | Survival rate (%) |
| Turfy soil | 75 |
| Turfy soil: perlite=1:1 | 85 |
| Turfy soil: perlite=3:1 | 90 |
As can be seen from the above table, turfy soil is used in the present application: a matrix regimen of perlite=3:1 can achieve a better survival rate.
In conclusion, the sterile rapid propagation method of trollius chinensis provided by the application adopts 75% alcohol and 4-6% sodium hypochlorite to disinfect trollius chinensis seeds, has a good disinfection effect and does not have adverse effects on the activity of explants and the environment. Furthermore, 20-30mg/L of cephalosporin and 20-40mg/L of mixed solution of 65-75% mancozeb and 70-80% chlorothalonil 1:1 are also added into the basic culture medium for disinfection, so that the pollution rate of seed germination can be further reduced, and the survival rate of seedlings is not influenced. And the phenomenon of secondary bacteria infection caused by endophyte generated by the trollius chinensis per se or incomplete sterilization can not occur in the subsequent experiments. The addition of the activated carbon and the vitamin C can play a role in synergy, not only can effectively improve browning, but also can avoid influencing the growth and differentiation of seedlings. The method can effectively solve the problems of poor disinfection effect, bacteria staining phenomenon and serious browning imagination of the trollius chinensis bunge, and is beneficial to improving the seedling rate of trollius chinensis bunge tissue culture.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (3)
1. The sterile rapid propagation method of trollius chinensis bunge is characterized by comprising the following steps of:
sterilizing the cleaned trollius chinensis seeds with 75% alcohol for 1-2min, and then sterilizing with 4-6% sodium hypochlorite for 20-30min to obtain sterile seeds;
Inoculating the sterile seeds into a germination culture medium for germination to obtain trollius chinensis germination seedlings; the germination medium is obtained by adding 20-30 mg/L of cephalosporin and 20-40mg/L of mixed solution into a basic medium, and then adding 40-60mg/L of activated carbon and 80-120mg/L of vitamin C, wherein the mixed solution is prepared by mixing 65-75% mancozeb and 70-80% chlorothalonil according to a ratio of 1:1; the basic culture medium is obtained by taking an MS culture medium as a base and adding +2mg/L6-BA+0.1 mg/L NAA, replacing sucrose in the MS culture medium with 23-27mg/L white sugar, and reducing the agar dosage to 5-6 g/L;
Artificially culturing the trollius chinensis bunge sprouting seedlings to obtain tissue culture seedlings, and hardening and transplanting the tissue culture seedlings; hardening and transplanting the tissue culture seedlings comprises the following steps: placing the tissue culture seedlings in the tissue culture bottle in advance for 5 days in a greenhouse for hardening seedlings, wherein the matrix is a mixture of turfy soil and perlite, and the turfy soil in the mixture is as follows: the volume ratio of the perlite is 3:1, sterilizing at high temperature in a sterilizing pot, regulating the pH value to 5.8, taking out the tissue culture seedlings from a tissue culture bottle, cleaning a culture medium on roots with water, soaking the culture medium in 1% carbendazim for 10min, and then planting the culture medium in a plug tray;
The artificial cultivation of the trollius chinensis bunge sprouting seedlings to obtain tissue culture seedlings comprises the following steps:
Transplanting the trollius chinensis bunge sprouting seedlings to a sterilized sprouting culture medium for continuous artificial sprouting culture to obtain trollius chinensis bunge aseptic seedlings, wherein the artificial sprouting culture is carried out for seven days in a dark culture mode, and then 12 h/12 h of light is carried out, 1300-1700Lux of light intensity is carried out, and the sprouting culture time is 28-30d;
Taking a stem segment of the trollius chinensis aseptic seedling as an explant, inserting the explant into a proliferation and secondary culture medium for proliferation and secondary culture, wherein the proliferation and secondary culture medium comprises MS culture medium +2mg/L6-BA +0.3-0.5mg/L NAA +40-60mg/L active carbon +80-120mg/L vitamin C, and the proliferation and secondary culture time is 58-62d; obtaining a cluster bud stem segment; inoculating the cluster bud stem sections into a rooting culture medium for culture, wherein the rooting culture medium is 1/2MS culture medium, 0.5mg/L NAA, 40-60mg/L active carbon and 80-120mg/L vitamin C, and the rooting culture time is 28-32d, so that the tissue culture seedlings are obtained.
2. The method of claim 1, wherein cleaning the trollius chinensis seeds comprises: wrapping the trollius chinensis seeds in sterilized gauze, soaking and cleaning in a detergent for 15-25min, and then washing with clear water for 25-35min.
3. A planting method of trollius chinensis bunge, which is characterized by comprising the sterile rapid propagation method of trollius chinensis bunge according to any one of claims 1-2.
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