CN117178890A

CN117178890A - Sterile seedling cultivation method for pseudo-ginseng

Info

Publication number: CN117178890A
Application number: CN202311251214.1A
Authority: CN
Inventors: 姚睿; 孙永灿; 张丽瑜; 卢凡; 江姗; 闫莉; 巫恬静
Original assignee: Zhejiang Findyou Biotechnology Co ltd
Current assignee: Zhejiang Findyou Biotechnology Co ltd
Priority date: 2023-09-26
Filing date: 2023-09-26
Publication date: 2023-12-08

Abstract

The application discloses a method for culturing aseptic seedlings of pseudo-ginseng, which comprises the following steps: and (3) disinfection: sterilizing Notoginseng radix seed with 75% alcohol for 1-3min, sterilizing with 13-17% sodium hypochlorite for 10-21min, and washing with sterile water to obtain sterilized seed; pollution screening: placing the sterilized seeds in a culture medium, observing for 7-12d, and screening to obtain pollution-free seeds; culturing: transferring the pollution-free seeds into a tissue culture bottle, culturing for 12-16d, taking out, transferring into the culture medium, continuously culturing for 1-12d, observing pollution condition, screening pollution-free plants, transferring into the tissue culture bottle again, and culturing to obtain the aseptic seedlings of the pseudo-ginseng. The cultivation method of the aseptic seedlings of the pseudo-ginseng is simple and nontoxic, and the aseptic seedlings of the pseudo-ginseng obtained by cultivation have extremely low pollution rate and higher germination rate, and can improve the yield and quality of the aseptic seedlings of the pseudo-ginseng.

Description

Sterile seedling cultivation method for pseudo-ginseng

Technical Field

The application relates to the field of pseudo-ginseng seedling cultivation, in particular to a cultivation method of pseudo-ginseng aseptic seedlings.

Background

Pseudo-ginseng is a precious traditional Chinese medicine and is widely applied to the field of traditional Chinese medicines. However, the supply of pseudo-ginseng cannot meet the market demand due to the poor reproductive capacity of pseudo-ginseng.

At present, in order to improve the yield and quality of pseudo-ginseng, tissue culture technology, aseptic culture technology and some genetic engineering technologies can be adopted to culture pseudo-ginseng, wherein the aseptic culture technology can effectively eliminate the pollution of pathogenic microorganisms, improve the growth vigor of pseudo-ginseng seedlings, reduce plant diseases and insect pests and improve the yield and quality of pseudo-ginseng.

However, the germination rate of the pseudo-ginseng seeds under aseptic culture is poor, and other components are added to easily destroy the aseptic environment of the aseptic seedlings, so that pollution is caused, and therefore, the low pollution rate and the high germination rate of the aseptic seedlings of the pseudo-ginseng are difficult to be simultaneously considered in the prior art, and the propagation of the aseptic culture of the pseudo-ginseng seedlings is difficult.

Disclosure of Invention

The application provides a method for culturing pseudo-ginseng aseptic seedlings, which aims to solve the problem that the pseudo-ginseng seedlings are difficult to reproduce under aseptic culture.

A method for culturing aseptic seedlings of radix Notoginseng comprises the following steps: and (3) disinfection: sterilizing Notoginseng radix seed with 75% alcohol for 1-3min, sterilizing with 5% -20% sodium hypochlorite for 10-21min, and washing with sterile water to obtain sterilized seed;

pollution screening: placing the sterilized seeds in a culture medium, observing for 7-12d, and screening to obtain pollution-free seeds;

culturing: transferring the pollution-free seeds into a tissue culture bottle, culturing for 12-16d, taking out, transferring into the culture medium, continuously culturing for 1-12d, observing pollution condition, screening pollution-free plants, transferring into the tissue culture bottle again, and culturing to obtain the aseptic seedlings of the pseudo-ginseng.

By adopting the technical scheme, the safety problem caused by adopting the mercuric chloride as the disinfectant in the traditional process and the germination influence on seeds are avoided. The method adopts a mode of combining alcohol disinfection and sodium hypochlorite disinfection to thoroughly kill microorganisms on the surfaces of the pseudo-ginseng seeds, adopts a culture medium in which seeds cannot germinate, screens pollution-free seeds in a mode of checking pollution conditions on the culture medium, reduces the pollution rate of final pseudo-ginseng aseptic seedlings, and improves the cultivation success rate of the pseudo-ginseng aseptic seedlings. In the culturing step, the seeds are continuously transferred into a culture medium for observing pollution after being cultured for a period of time, then the pollution-free continuous culture is screened, pollution-free aseptic seedlings are further screened at key nodes of seed germination, and the pollution of the aseptic seedlings can be greatly improved and reduced by two times of screening.

In addition, after the disinfection by adopting alcohol and sodium hypochlorite, the seeds are washed by sterile water, so that on one hand, the influence of residual alcohol and sodium hypochlorite on the subsequent culture of the seeds is reduced; on the other hand, the water content of the seeds after the sterile water washing is improved, and the air drying and inactivation of the seeds caused by the volatilization of the water in the pollution screening step can be reduced.

Preferably, the sterilization process includes the steps of:

and (3) disinfection: taking pseudo-ginseng seeds, removing seed coats, sterilizing with 75% alcohol for 1-3min, sterilizing with 13-17% sodium hypochlorite for 10-21min, and washing with sterile water to obtain sterilized seeds.

By adopting the technical scheme, the seed coats of the pseudo-ginseng seeds are removed, so that a large number of microorganisms contained in the uneven surfaces of the seed coats can be removed, and the seed disinfection effect is further improved. In addition, the seed coats are removed before the combination of alcohol and sodium hypochlorite is adopted for sterilization, so that microorganisms brought in the process of removing the seed coats can be sterilized, and the sterility of seeds in the subsequent culture process is ensured.

Preferably, the sterilization process includes the steps of:

and (3) disinfection: soaking Notoginseng radix seeds in a first antibacterial agent for 0.5-1 hr, taking out, sterilizing with 75% alcohol for 1-3min, sterilizing with 5-20% sodium hypochlorite for 10-21min, and washing with sterile water to obtain sterilized seeds;

the first antibacterial agent adopts 3-7% carbendazim solution.

By adopting the technical scheme, the carbendazim can inhibit the processes of cell division and nuclear division of pathogenic bacteria, and improve the sterility of seeds. The method adopts a soaking mode, further limits the concentration and soaking time of the carbendazim, reduces the influence of the antibacterial agent on the subsequent culture of the seeds, improves the sterilization effect on the seeds, and improves the sterility of the seeds in a synergistic way through soaking the first antibacterial agent, alcohol sterilization and sodium hypochlorite sterilization.

Preferably, the method for cultivating the aseptic seedlings of the pseudo-ginseng comprises the following steps:

and (3) disinfection: taking pseudo-ginseng seeds, removing seed coats, sterilizing with 75% alcohol for 1-3min, sterilizing with 13-17% sodium hypochlorite for 10-21min, and washing with sterile water to obtain sterilized seeds;

excision: cutting 70-76% cotyledon from pollution-free seed to obtain seed embryo with 24-30% cotyledon;

culturing: transferring the embryo into a tissue culture bottle, culturing for 12-16d, transferring into the culture medium, continuously culturing for 1-12d, observing pollution, screening pollution-free plants, transferring into the tissue culture bottle again, and culturing to obtain the sterile seedling of radix Notoginseng.

By adopting the technical scheme, before the cotyledons are transferred into the tissue culture bottle for culture, part of the cotyledons are cut off, so that the nutrition requirement in the process can be reduced, the energy and nutrition waste in the aseptic culture process can be reduced, and the emphasis is that the aseptic culture is adopted, if nutrient substances are required to be added again due to insufficient nutrition, new microorganisms are likely to be introduced, and pollution is caused. In addition, the volume occupied by seeds in the tissue culture bottle can be reduced, and the influence of contact inhibition on the growth of the seeds can be reduced.

The partial cotyledon is cut off, so that microorganisms contained in the cotyledon can be reduced, and pollution is reduced. In addition, the excision step is selected between the pollution screening and culturing steps, so that the influence of the excision step, the sterilization step and the sterilization step on the embryo can be avoided, and the pollution possibly introduced in the excision step can be reduced because the observation of one-time pollution is also carried out in the culturing step.

And part of cotyledons are cut off, the differentiation trend of seeds can be influenced, the root system part is grown first, and the breeding quality of the sterile seedlings is improved.

And part of cotyledons are cut off, so that the growth state of the seeds in the culture process is easier to observe, the screening accuracy is improved, and the sterility is improved.

Preferably, the medium is a solid medium without hormones.

Typically, but not by way of limitation, the solid medium is selected from one of MS solid medium, BS solid medium or WPM solid medium.

By adopting the technical scheme, the solid culture medium without hormone ensures that seeds do not germinate in the pollution screening step, so that the pollution condition of the sterilized seeds is easier to observe. On the other hand, after screening, the seeds are cultivated, and proper seeds are selected for germination, so that the cultivation cost of aseptic seedlings can be reduced, and the pollution of the aseptic seedlings can be reduced.

Preferably, the tissue culture flask comprises a base material, wherein the base material comprises a composition of one or more of cotton, fine river sand or vermiculite, and the water content of the base material is 10-25%.

By adopting the technical scheme, the culture is carried out by adopting the base material with the water content of 10-25%, wherein the base material adopts cotton, river sand or vermiculite, the materials have better asepsis, air permeability and drainage, the rot or dryness of the pseudo-ginseng seeds can be reduced, the germination rate is improved, and the water is not required to be added in the culture process by adopting the materials, so that the introduction of new pollutants is reduced, and the pollution is reduced.

Preferably, the tissue culture bottle is sterilized at high temperature, and the high temperature sterilization comprises the following steps:

sealing the tissue culture bottle, sterilizing at 121 deg.C and 1.05-1.1MPa for 45-75min to obtain the high temperature sterilized tissue culture bottle.

By adopting the technical scheme, the tissue culture bottle after high-temperature sterilization can provide better sterility, reduce the pollution of aseptic seedlings in the cultivation process, decompose certain components in the base materials, provide more nutritional components and improve the germination rate of the aseptic seedlings.

Preferably, the tissue culture bottle further comprises a second antibacterial agent, and the weight ratio of the second antibacterial agent to the base material is (0.1-0.3): 100, the second antibacterial agent adopts a plant tissue culture antibacterial agent.

By adopting the technical scheme, the second antibacterial agent is added into the tissue culture bottle, so that the sterility of the tissue culture bottle is further improved, and the antibacterial effect can be generated from different aspects in the seed cultivation process.

In addition, the second antibacterial agent adopts a plant tissue culture antibacterial agent, and the plant tissue culture antibacterial agent has a good heat-resistant effect, can not be decomposed and disabled along with the high-temperature sterilization process, so that the possibility that the microorganism is introduced by adding the plant tissue culture antibacterial agent into the tissue culture bottle after the high-temperature sterilization is reduced, and the sterility of the whole cultivation method is further improved.

Preferably, in the culturing step, the culturing condition of the tissue culture bottle is 20-25 ℃,1400-1800LX.

By adopting the culture conditions, the germination rate of the seeds is higher, and the growth condition of the aseptic seedlings is also better.

Preferably, the environment of the contamination screening step includes the following parameters: the temperature is 15-25 ℃ and the environment is dark.

By adopting the culture conditions, the germination of the seeds can be reduced, the damage of environmental factors to the seeds is reduced, and the germination rate of the subsequently cultivated seeds is improved.

In summary, the application has the following beneficial effects:

1. in the disinfection step, the seed coats are removed, and the disinfection is carried out by matching with alcohol and sodium hypochlorite, so that microorganisms in seeds can be greatly reduced, and compared with the scheme of adopting mercuric chloride for disinfection in the prior art, the safety is improved, and meanwhile, the sterility is ensured. In addition, the germination rate and sterility of the seeds can be improved by cutting off part of the cotyledons after the pollution screening step. Namely, the sterility and germination rate of the aseptic seedlings of the pseudo-ginseng are improved by adopting a mode of removing seed coats and part of cotyledons.

2. The culture medium adopts river sand or vermiculite with the water content of 10-25% to replace the traditional quartz sand culture medium, so that the need of supplementing water into the tissue culture bottle in the culture process can be avoided, the externally introduced microorganisms are reduced, the sterility of the tissue culture bottle is improved, and the culture medium has good water drainage and air permeability, and the seed rot or dryness is avoided under the condition of guaranteeing the seed germination humidity.

Drawings

FIG. 1 shows the growth of aseptic seedlings of Notoginseng radix in the cultivation step of example 1.

FIG. 2 is the contamination of seeds in the contamination screening step of example 1.

FIG. 3 shows the growth of the sterile seedlings of Panax notoginseng in the cultivation step of example 2.

FIG. 4 is a sample of seed contamination in the contamination screening step of example 3.

FIG. 5 shows the growth of pseudo-ginseng seedlings in the cultivation step of example 3, and the pseudo-ginseng seedlings are transferred into a culture medium to observe pollution.

FIG. 6 is a case where seeds were contaminated in the contamination screening step in comparative example 1.

FIG. 7 shows the growth of pseudo-ginseng seedlings in a medium during the cultivation step in comparative example 1.

FIG. 8 shows the growth of pseudo-ginseng seedlings in a medium during the cultivation step in comparative example 2.

Detailed Description

In order to make the objects, technical solutions and advantageous technical effects of the present application more clear, the present application will be described in detail below. It should be noted that the various aspects, features, embodiments, and advantages thereof described herein may be compatible and/or may be combined together.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

The application relates to a method for culturing sterile seedlings of pseudo-ginseng.

The present application will be specifically described below.

In the prior art, the biological material is generally disinfected by adopting the mercuric chloride, but the mercuric chloride belongs to a highly toxic substance, so that a series of personal safety problems are easily caused, and in addition, in the cultivation process, the external substances such as an antibacterial agent or water are additionally added to introduce pollutants, so that the sterility is influenced. In addition, the aseptic seedlings are always found to be polluted in the seedling emergence stage in the prior art, which is equivalent to the whole waste of work and materials before cultivation, and the whole cultivation period is prolonged, so that the cultivation efficiency is very low.

The method for cultivating the sterile seedlings of the pseudo-ginseng adopts non-toxic disinfection treatment, combines special treatment modes such as seed coat excision, cotyledon excision and the like to improve the sterility of seeds, and adds a pollution screening step before and during cultivation, so that the pollution condition can be found as early as possible and corresponding screening can be carried out.

In some embodiments, in the step of sterilizing, the time of sterilizing the alcohol may be 1,2,3 minutes or any two of the above values, the concentration of sodium hypochlorite may be 13%,14%,15%,16%,17% or any two of the above values, and the time of sterilizing the sodium hypochlorite may be 10,15,21 minutes or any two of the above values.

In the contamination screening step, the time of observation may be selected to be 7,8,9,10,11,12d or within a range consisting of any two of the above values. The temperature of the culture may be selected from the range of 15 ℃,20 ℃,25 ℃ or any two of the above values.

In the culturing step, the water content of the base material in the tissue culture flask may be selected to be 10%,15%,20%,25% or any two of the above values, the culturing time in the tissue culture flask for the first time may be selected to be 12, 13,14,15,16d or any two of the above values, the culturing time in the medium may be selected to be 1,2,3,4,5,6,7,8,9,10,11,12d or any two of the above values, and the culturing time in the tissue culture flask for the second time may be selected to be 7,14,20d or any two of the above values. The culture conditions in the tissue culture flask may be 20℃and 25℃or a range consisting of the above two values, and the illumination intensity may be selected from 1400LX, 630 LX or 180 LX or a range consisting of any two of the above values. The amount of the second antimicrobial agent added to the tissue culture flask may be 0.1%,0.2%,0.3% or any two of the above values by mass of the base material.

Examples

Example 1, a method for culturing aseptic seedlings of pseudo-ginseng, comprising the following steps:

and (3) disinfection: sterilizing Notoginseng radix seed with 75% alcohol, mixing for 3min, sterilizing with 15% NaClO for 21min, and drying with clean filter paper to obtain sterilized seed;

pollution screening: transferring the sterilized seeds into an MSO (MS solid culture medium without hormone) culture medium, culturing at 20deg.C in dark, observing pollution condition of the seeds after 7 days, and screening to obtain pollution-free seeds;

culturing: transferring the pollution-free seeds into a tissue culture bottle, culturing for 14d at 20 ℃ in a dark way, taking out and transferring the seeds into an MSO culture medium for culturing for 7d; and screening pollution-free pseudo-ginseng seedlings, inoculating the pseudo-ginseng seedlings back to a tissue culture bottle, and culturing the pseudo-ginseng seedlings at 22 ℃ and 1500LX light intensity to obtain the pseudo-ginseng aseptic seedlings.

Wherein, the base material in the tissue culture bottle adopts fine river sand containing 20 percent of sterile water (the thickness of the river sand is 2-4 cm, the specification of the tissue culture bottle is 8-10 cm in diameter and 10-15 cm in height). The base material in the tissue culture bottle is subjected to high-pressure sterilization, and the high-pressure sterilization step adopts the following steps: sealing the tissue culture bottle filled with the base material, sterilizing at 121 ℃ and 1.05-1.1MPa for 45-75min to obtain the tissue culture bottle after high-temperature sterilization.

Example 2, a method for culturing aseptic seedlings of pseudo-ginseng, comprises the following steps:

and (3) disinfection: soaking Notoginseng radix seeds in 5% carbendazim solution for 30min, washing the seeds with tap water, sterilizing with 75% alcohol, mixing for 3min, sterilizing with 15% NaClO for 25min, and sterilizing to obtain sterilized seeds;

Example 3, a method for culturing aseptic seedlings of pseudo-ginseng, comprises the following steps:

and (3) disinfection: taking pseudo-ginseng seeds, washing the seeds cleanly by using tap water, gently peeling the seed coats by using forceps, sterilizing the seeds by using 75% alcohol, mixing the seeds uniformly by upside down for 3min, sterilizing the seeds by using 15% NaClO, and upside down for 25min to obtain the sterilized seeds;

Example 4, a method for culturing aseptic seedlings of pseudo-ginseng, comprising the following steps:

and (3) disinfection: taking pseudo-ginseng seeds, washing the seeds cleanly by using tap water, gently peeling the seed coats by using forceps, soaking the seeds for 10min by using 5% carbendazim, sterilizing the seeds by using 75% alcohol, mixing the seeds uniformly by upside down for 3min, sterilizing the seeds by using 15% NaClO, and reversing the seeds by upside down for 25min to obtain the sterilized seeds;

Example 5, a cultivation method of aseptic seedlings of pseudo-ginseng is different from example 2 in that the tissue culture bottle further comprises a plant tissue culture antibacterial agent, and the addition amount of the plant tissue culture antibacterial agent is 0.2% of the total mass of the base material.

Example 6, a method for cultivating aseptic seedlings of pseudo-ginseng, comprising the steps of:

excision: taking pollution-free seeds, and cutting 74% of cotyledons to obtain seed embryos with 26% of cotyledons;

Wherein, in the excision step, the excision amount of the cotyledon is selected to be between 70 and 76%. The tissue culture bottle comprises a composition of a base material containing 20% of sterile water and a plant tissue culture antibacterial agent, wherein the addition amount of the plant tissue culture antibacterial agent is 0.2% of the total mass of the base material. The tissue culture bottle is subjected to high-pressure sterilization, and the high-pressure sterilization step comprises the following steps: sealing the tissue culture bottle filled with the base material, sterilizing at 121 ℃ and 1.05-1.1MPa for 45-75min to obtain the tissue culture bottle after high-temperature sterilization.

Comparative example

Comparative example 1, a method of culturing a sterile seedling of Panax notoginseng, is different from example 1 in that the concentration of sodium hypochlorite is 10%.

Comparative example 2, a method of raising sterile seedlings of pseudo-ginseng, differs from example 1 in that the concentration of sodium hypochlorite is 20%.

Comparative example 3, a method for aseptic seedling cultivation of pseudo-ginseng, is different from example 1 in that the sterilization treatment adopts the following steps: sterilizing Notoginseng radix seed with 15% NaClO, reversing for 21min, and drying the sterilized seed with clean filter paper to obtain sterilized seed.

Comparative example 4, a method for culturing aseptic seedlings of pseudo-ginseng, adopts the following steps:

and (3) disinfection: taking pseudo-ginseng seeds, removing peel, soaking for 12 hours with 300mg/L gibberellin, washing for several times with tap water, then washing for 30 minutes with running water, sterilizing for 30 seconds with 75% alcohol, and washing for 2-3 times with sterile water. Sterilizing with 0.1% mercuric chloride for 8-10 min, washing with sterile water for 3-5 times, sucking water with sterile paper, and inoculating into sterilized MS solid culture medium for culturing.

Comparative example 5, a method for culturing aseptic seedlings of pseudo-ginseng, adopts the following steps:

removing outer red peel of Notoginseng radix seed after harvesting, sterilizing with 3% sodium hypochlorite solution for 5min, repeatedly washing with distilled water, mixing with wet river sand, and storing in shade for after-ripening. And respectively placing the quartz sand and the culture dish into a sterilizing pot for high-temperature sterilization for 2 hours, and placing the quartz sand according to 60 g/dish for standby. In spring, healthy and full seeds of pseudo-ginseng with consistent size are selected and sown in sterilized culture dishes (diameter phi=90 mm), 25 seeds of each dish are covered, water is added once in a period of 3d, and the sterile seedlings of pseudo-ginseng are obtained.

Performance test

Based on 1000 seeds which enter the cultivation step, the cultivation methods of examples 1-5 and comparative examples 1-5 were observed for the pollution rate and germination rate of the obtained sterile seedlings of pseudo-ginseng, and 6 groups of parallel tests were performed for each group of methods, and the average value was taken, and the results are shown in Table 1.

Table 1: results of examples 1 to 6, comparative examples 1 to 5

By combining examples 1-6 and comparative examples 1-5 and combining table 1, the optimized cultivation method of the aseptic seedlings of the pseudo-ginseng can greatly reduce the pollution rate of the aseptic seedlings of the pseudo-ginseng, improve the germination rate to more than 80%, has good popularization prospect, and is characterized in that the seed coats are removed, part of cotyledons are removed, harmful microorganisms carried by the seeds are greatly reduced, and the existence of harmful microorganisms in the environment is further reduced by the cooperation of multiple disinfection means such as alcohol, sodium hypochlorite, a first antibacterial agent, a second antibacterial agent and the like, so that the pollution rate of the aseptic seedlings of the pseudo-ginseng obtained by cultivation is reduced to the minimum. In addition, partial cotyledon is cut off, and fine river sand with corresponding water content is creatively selected as a base material in the tissue culture bottle, so that the introduction of harmful microorganisms inside and outside can be reduced, and the germination rate of seeds can be improved.

In combination with examples 1-4, comparative examples 1-3 and Table 1, it can be seen that the contamination rate of the aseptic seedlings of Panax notoginseng can be further reduced by immersing the first antibacterial agent after removing the seed coats, because the uneven surfaces of the seed coats contain a large amount of microorganisms, which can affect the effect of other sterilization treatments.

As can be seen from the combination of examples 5-6 and Table 1, the operation of cutting the cotyledons greatly reduces the harmful microorganisms contained in the aseptic seedlings of pseudo-ginseng to reduce the contamination rate, and in addition, the germination rate of the aseptic seedlings of pseudo-ginseng is greatly improved, because the cutting of part of the cotyledons can reduce the nutrition demand in the aseptic culture, and the waste of energy and nutrition in the aseptic culture is reduced, and it is emphasized that, due to the aseptic culture, if the nutrient shortage is caused, new microorganisms are likely to be introduced because of the need of re-adding the nutrient substances, resulting in contamination. The volume occupied by seeds in the tissue culture bottle can be reduced, and the influence of contact inhibition on the growth of the seeds can be reduced. The partial cotyledon is cut off, so that microorganisms contained in the cotyledon can be reduced, and pollution is reduced. In addition, the excision step is selected between the pollution screening and culturing steps, so that the influence of the excision step, the sterilization step and the sterilization step on the embryo can be avoided, and the pollution possibly introduced in the excision step can be reduced because the observation of one-time pollution is also carried out in the culturing step. And part of cotyledons are cut off, the differentiation trend of seeds can be influenced, the root system part is grown first, and the breeding quality of the sterile seedlings is improved.

In combination with example 1, comparative examples 1-3 and Table 1, it can be seen that the requirements of the sterile seedlings of Panax notoginseng for the sterilization treatment are very high, and the fine adjustment of any parameter may have a great influence on the contamination rate and germination rate of the sterile seedlings.

As can be seen by combining examples 1-6 and comparative example 4 with Table 1, the cultivation method of the application has no great difference in the final contamination rate compared with the conventional mercuric oxide disinfection method, even can achieve lower contamination rate than the conventional mercuric oxide disinfection method by adding the steps of contamination screening, seed coat removal, cotyledon removal and the like, and in addition, the cultivation method of the application overcomes the obstacle and can achieve the germination rate of more than 80 because the mercuric oxide disinfection method is harmful to human bodies and affects the germination rate of seeds to a certain extent, thus being a great progress for cultivation of aseptic seedlings of pseudo-ginseng.

As can be seen from the combination of example 1, comparative example 5 and Table 1, the prior art has a process of removing the seed coats, but the pollution rate cannot be well reduced by removing the seed coats alone, the germination rate of the seeds obtained by the method is low, and in addition, the traditional quartz sand is used as the base material, so that the pollution rate is high, and the influence on the germination rate of the seeds is also large. The cultivation technology is characterized in that the center is not placed on a single technology means for removing seed coats, the seed coats are removed, partial cotyledons are removed, and a plurality of disinfection means are cooperated to take effect on the single technology means for removing the seed coats and other technology means, so that the effect of 1+1 is achieved, and the cultivation method of the pseudo-ginseng aseptic seedlings with extremely low pollution rate and germination rate of more than 90% is finally obtained. Similarly, the fine river sand with the water content of 10-25% is used for replacing quartz sand, so that the three pseudo-ginseng seeds with the characteristics of humidity, temperature and air permeability are dependent on germination, and no additional water is needed in the germination process, so that the introduction of harmful microorganisms is reduced, and the germination rate is improved.

The present embodiment is only for explanation of the present application and is not to be construed as limiting the present application, and modifications to the present embodiment, which may not creatively contribute to the present application as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present application.

Claims

1. A method for culturing the aseptic seedlings of notoginseng is characterized by comprising the following steps

And (3) disinfection: sterilizing Notoginseng radix seed with 75% alcohol for 1-3min, sterilizing with 13-17% sodium hypochlorite for 10-21min, and washing with sterile water to obtain sterilized seed;

2. The method for culturing sterile seedlings of pseudo-ginseng according to claim 1, wherein the sterilization treatment comprises the steps of:

3. The method for culturing sterile seedlings of pseudo-ginseng according to claim 1, wherein the sterilization treatment comprises the steps of:

the first antibacterial agent adopts 3-7% carbendazim solution.

4. The method for culturing the sterile seedlings of pseudo-ginseng according to claim 1, comprising the following steps of

5. The method for culturing sterile seedlings of pseudo-ginseng according to claim 1, wherein the culture medium is a solid culture medium without hormone.

6. The method of claim 1, wherein the tissue culture flask comprises a base material comprising a composition of one or more of cotton, fine river sand, or vermiculite, and wherein the water content of the base material is 10-25%.

7. The method for culturing sterile seedlings of pseudo-ginseng according to claim 6, wherein the tissue culture bottle is sterilized at high temperature, and the high temperature sterilization comprises the following steps:

8. The method for culturing sterile seedlings of pseudo-ginseng according to claim 7, wherein the tissue culture bottle further comprises a second antibacterial agent, and the weight ratio of the second antibacterial agent to the base material is (0.1-0.3): 100, the second antibacterial agent adopts a plant tissue culture antibacterial agent.

9. The method according to claim 1, wherein the culturing conditions in the tissue culture flask are 20-25 ℃,1400-1800LX.

10. The method of claim 1, wherein the environment of the contamination screening step comprises the following parameters: the temperature is 15-25 ℃ and the environment is dark.