CN116250480B - A kind of aseptic rapid propagation method of nasturtium and planting method of nasturtium - Google Patents

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

A kind of aseptic rapid propagation method of nasturtium and planting method of nasturtium

Technical Field

The invention relates to the technical field of nasturtium cultivation, and in particular to a method for aseptic rapid propagation of nasturtium and a method for planting nasturtium.

Background technique

Nasturtium is a perennial plant of the Ranunculaceae family. Its flowers have extremely high medicinal value and can be used as medicine or tea. In recent years, as people have become more aware of the medicinal and ornamental value of nasturtium, the number of nasturtiums picked has increased year by year, seriously damaging the wild nasturtium resources. Therefore, it is of great significance to introduce, domesticate and artificially cultivate wild nasturtiums.

The sexual reproduction of nasturtium generally has problems such as low germination rate and poor survival rate of seedlings. Tissue culture technology is an effective way to conserve and develop plant resources. It can preserve plant germplasm resources in vitro through tissues and organs, and can also reduce the demand for wild resources by providing a large number of seedlings through factory-based seedling cultivation.

Most of the existing nasturtium tissue culture technologies use 0.1% mercuric chloride to sterilize explants, but mercuric chloride is a highly toxic agent, and the residue is not easy to remove, which is lethal to the activity of explants and causes serious damage to the environment. When using the nasturtium callus induction approach for tissue culture, the callus rate is low, the browning is serious, and the callus redifferentiation is difficult. The regeneration system is not stable enough and it is difficult to induce a complete plant. When using the nasturtium sterile seed rapid propagation approach for tissue culture, most of them consider the sterilization effect of the sterilizer on the seeds, but ignore the effect of the sterilizer on the activity of the explants, resulting in a low seed seedling rate. At the same time, the browning phenomenon accompanying the germination and proliferation culture process is ignored. In addition, the tissue culture of nasturtium is currently only carried out in the laboratory stage, which is costly and the tissue culture system is not optimized.

In view of this, the present invention is proposed.

Summary of the invention

The purpose of the present invention is to provide a method for aseptic rapid propagation of nasturtium, which can reduce the contamination rate during seed germination and alleviate the problem of seedling browning, thereby achieving good cultivation effect at a relatively low cost.

The invention aims to provide a method for planting nasturtium.

The present invention is achieved in that:

In a first aspect, the present invention provides a method for aseptic rapid propagation of nasturtium, comprising:

The cleaned nasturtium seeds are sterilized with 75% alcohol for 1-2 minutes, and then sterilized with 4-6% sodium hypochlorite for 20-30 minutes to obtain sterile seeds;

The sterile seeds are inoculated into a germination medium for germination to obtain nasturtium germination seedlings; wherein the germination medium is obtained by first adding 20-30 mg/L of cephalosporin and 20-40 mg/L of a 1:1 mixture of 65-75% mancozeb and 70-80% thiophanate-methyl to a basic culture medium, and then adding 40-60 mg/L of activated carbon and 80-120 mg/L of vitamin C;

The nasturtium germination seedlings are artificially cultivated to obtain tissue culture seedlings, and the tissue culture seedlings are hardened and transplanted.

In an optional embodiment, the basal culture medium is based on MS culture medium, wherein sucrose in the MS culture medium is replaced with 23-27 mg/L of white sugar, and the amount of agar is reduced to 5-6 g/L.

In an optional embodiment, the basal culture medium is further supplemented with 1.0-2.5 mg/L 6-BA and 0.1-0.5 mg/L NAA.

In an optional embodiment, artificially cultivating the nasturtium germination seedlings to obtain tissue culture seedlings comprises:

Transplanting the nasturtium germination seedlings into a sterilized germination medium for further artificial germination culture to obtain nasturtium sterile seedlings, wherein the artificial germination culture is first dark culture for seven days, followed by 12 hours of light/12 hours of darkness, with a light intensity of 1300-1700 Lux, and a germination culture time of 28-30 days;

The stem segments of the nasturtium sterile seedlings are used as explants, and the explants are inserted into a proliferation and subculture medium for proliferation and subculture to obtain clustered bud stem segments; the clustered bud stem segments are inoculated into a rooting medium for culture to obtain tissue culture seedlings.

In an optional embodiment, the proliferation and subculture medium is obtained by adding 1.5-2.5 mg/L 6-BA and 0.3-0.4 mg/L NAA to MS medium, and then adding 40-60 mg/L activated carbon and 80-120 mg/L vitamin C, and the proliferation and subculture time is 58-62 days.

In an optional embodiment, the rooting medium is obtained by adding 0.1-0.5 mg/L NAA, 40-60 mg/L activated carbon and 80-120 mg/L vitamin C to 1/2 MS medium, and the rooting culture time is 28-32 days.

In an optional embodiment, hardening and transplanting the tissue culture seedlings includes: placing the tissue culture seedlings in the tissue culture bottle in a greenhouse for hardening 5 days in advance, the substrate is a mixture of peat soil and perlite, the volume ratio of peat soil: perlite in the mixture is 3:1, high-temperature sterilization is performed in a sterilizing pot, the pH value is adjusted to 5.8, and then the tissue culture seedlings are taken out from the tissue culture bottle, the culture medium on the roots is cleaned with water, soaked in 1% carbendazim for 10 minutes, and then planted in a plug tray.

In an optional embodiment, cleaning the nasturtium seeds includes: wrapping the nasturtium seeds in sterilized gauze, soaking and cleaning in detergent for 15-25 minutes, and then rinsing with clean water for 25-35 minutes.

In a second aspect, the present invention provides a method for planting nasturtium, which comprises the aseptic rapid propagation method of nasturtium described in any one of the aforementioned embodiments.

The present invention has the following beneficial effects:

The aseptic rapid propagation method of nasturtium provided in the present application uses 75% alcohol + 2-10% sodium hypochlorite to disinfect nasturtium seeds, which has good disinfection effect and will not have adverse effects on explant activity and the environment. Further, in the present application, 20-30mg/L of cephalosporin and 20-40mg/L of 65-75% mancozeb and 70-80% thiophanate-methyl 1:1 mixed solution are added to the basal culture medium for disinfection, which can further reduce the contamination rate of seed germination and will not affect the survival rate of seedlings. And in subsequent experiments, there will be no re-infection phenomenon due to the production of endophytes by the nasturtium itself or incomplete disinfection. The addition of activated carbon and vitamin C can play a synergistic role, which can not only effectively improve browning, but also avoid affecting the growth and differentiation of seedlings. In the present application, the above-mentioned treatment can effectively improve the poor disinfection effect of nasturtium, the serious problems of bacterial infection and browning imagination, which is conducive to improving the seedling rate of nasturtium tissue culture. The present application uses the aseptic germination and rapid propagation method of nasturtium seeds to aseptically and rapidly propagate, and the obtained offspring of the same seedling single plant system are easy to establish, the propagation steps are simplified, and the effective propagation rate is high, which is of great significance for preserving and expanding the nasturtium population. The nasturtium bred by the aseptic germination and rapid propagation method of nasturtium seeds in the present application has a seed germination rate of 80.67% after 30 days, a proliferation coefficient of 4.34 within 60 days, and a transplant survival rate of more than 90%, which greatly improves the propagation coefficient of nasturtium and provides a very effective propagation method for the introduction and domestication, preservation, gardening and horticulture of the species and the utilization of its scientific research value.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

FIG1 is a schematic diagram of the germination of nasturtium provided in Example 1 of the present application;

FIG2 is a schematic diagram of the artificial cultivation of nasturtium to form sterile seedlings provided in Example 1 of the present application;

FIG3 is a schematic diagram of the tissue culture propagation of nasturtium provided in Example 1 of the present application;

FIG4 is a schematic diagram of the rooting of trollius tissue culture provided in Example 1 of the present application.

Detailed ways

In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

The present invention provides a method for aseptic rapid propagation of nasturtium, which comprises the following steps:

S1. Obtain sterile seeds.

Wrap the nasturtium seeds in sterilized gauze to effectively reduce seed leakage during shaking. Soak and clean them in detergent for 15-25 minutes, then rinse with clean water for 25-35 minutes.

The cleaned golden lotus seeds are moved to a clean bench, disinfected with 75% alcohol for 1-2 minutes, and then disinfected with 2-10% sodium hypochlorite for 20-30 minutes to obtain sterile seeds. It has been found that the existing golden lotus seed disinfection uses 0.1% mercuric chloride to sterilize the explants, which reduces the contamination rate but also affects the survival rate of the seeds. In this application, the combination of 75% alcohol and 2-10% sodium hypochlorite is used to disinfect and sterilize in sequence, which has the best effect and will not have adverse effects on the activity of the explants and the environment. In this embodiment, the main purpose of 75% alcohol is not for disinfection, but for wetting the surface of the plant; sodium hypochlorite is the main disinfectant. 75% alcohol is to allow sodium hypochlorite to act better on the surface of the seeds. When the alcohol concentration is too high or too low, the disinfection and sterilization effect is poor. Similarly, too high or too low a concentration of sodium hypochlorite is not conducive to disinfection and sterilization.

S2. Inoculate the sterile seeds into a germination medium for germination to obtain nasturtium germination seedlings.

In the present application, the basal medium is based on the MS medium, the sucrose in the MS medium is replaced with 23-27 mg/L of white sugar, and the amount of agar is reduced to 5-6 g/L. Among them, the specific components of the MS medium are known and will not be described in detail in the present application.

The germination medium is prepared by first adding 20-30 mg/L of cephalosporin and 20-40 mg/L of a 1:1 mixture of 65-75% mancozeb and 70-80% thiophanate-methyl to the basic medium, and after disinfection, adding 40-60 mg/L activated carbon and 80-120 mg/L vitamin C to the basic medium to obtain the germination medium of the present application.

Normally, the culture medium can be inoculated directly after conventional disinfection (such as high temperature disinfection). However, in this application, 20-30 mg/L of cephalosporin and 20-40 mg/L of 65-75% mancozeb and 70-80% thiophanate-methyl 1:1 mixture are added to the basal culture medium on the basis of conventional disinfection, which can further reduce the contamination rate of seed germination and will not affect the survival rate of seedlings. In addition, in subsequent experiments, there will be no re-infection due to the production of endophytes by the nasturtium itself or incomplete disinfection. However, the existing nasturtium tissue culture process is accompanied by bacterial contamination.

Nasturtium contains polyphenol compounds. The trauma when cutting the buds will activate the polyphenol oxidase in the tissue, oxidize the polyphenols into brown quinones, and cause browning at the incision of the explant. It will penetrate into the culture medium and brown the culture medium, which will seriously affect the growth and differentiation of the culture and even cause the death of the culture. In this application, the addition of activated carbon and vitamin C can improve the problem of browning of some seedlings during the culture process. Among them, activated carbon can adsorb brown quinones that have an inhibitory effect on growth, and vitamin C can effectively prevent browning as an oxidant. In this application, activated carbon and vitamin C are used in combination, which can play a synergistic role, not only effectively improving browning, but also avoiding affecting the growth and differentiation of seedlings.

It should be understood that although proliferation and subculture cluster bud induction are more prone to browning, browning may occur in all experimental processes. Therefore, after screening out the best solution to prevent browning, the present application also added activated carbon and vitamin C to all culture media. Further, in the present application, 1.5-2.5 mg/L 6-BA and 0.1-0.2 mg/L NAA are also added to the basal culture medium. Adding the above growth hormones during the sterile seed germination process can promote the germination rate, with the highest germination rate of 80.67%.

S3. Artificially cultivate the nasturtium germination seedlings to obtain tissue culture seedlings.

(1) Transplanting the nasturtium seedlings into a sterilized optimal germination medium, inoculating 6-8 nasturtium seedlings per bottle, and artificially culturing them in an artificial culture room at a temperature of 25±2° C., a humidity of 70%, and a photoperiod of 12 h light/12 h dark, to obtain nasturtium sterile seedlings;

(2) On sterile filter paper, leaves are removed with a scalpel and cut into stem segments containing axillary bud nodes, and the stem segments of the sterile nasturtium seedlings are used as explants. The explants are inserted into a proliferation and subculture medium in an artificial culture room under the following culture conditions: temperature 25±2°C, humidity 70%, and photoperiod 12 h light/12 h dark, to obtain clustered bud stem segments; wherein the proliferation and subculture medium is obtained by adding 1.5-2.5 mg/L 6-BA and 0.3-0.4 mg/L NAA, and then adding 40-60 mg/L activated carbon and 80-120 mg/L vitamin C to MS medium.

(3) inoculating the clustered bud stem segments into a rooting medium to obtain tissue culture seedlings, wherein the rooting medium is obtained by adding 0.4-0.6 mg/L NAA, 40-60 mg/L activated carbon and 80-120 mg/L vitamin C to 1/2 MS medium.

S4. Harden and transplant the tissue culture seedlings.

Before transplanting the golden lotus tissue culture seedlings, hardening the seedlings can improve the resistance of the tissue culture seedlings to adverse environments and improve the survival rate of the seedlings after transplanting. After the golden lotus tissue culture seedlings take root for 30 days, transfer the tissue culture seedlings to the greenhouse for natural light exposure (avoid direct sunlight). The indoor temperature should be close to the temperature of the culture room (23℃-25℃), so that the seedlings can slowly adapt to the external environmental conditions to improve the adaptability of the golden lotus tissue culture seedlings to the external environment. Half open the bottle cap after 2 days, add a little sterile water to cover the surface of the culture medium, leave it for two days, completely open the bottle cap after 3 days, and transplant it after 3d-5d. Then take out the golden lotus tissue culture seedlings from the tissue culture bottle, clean the culture medium on the roots with water to prevent bacteria and other diseases. Do not hurt the root system when cleaning, then soak it in 1% carbendazim for 10 minutes, and then plant it in a plug tray. Make a small hole in the middle of the hole in the plug tray, insert the root system, cover the root system with a mixture of peat soil and perlite in a ratio of 3:1 (sterilized at high temperature in a sterilizer), and water thoroughly. When transplanting, plant one seedling in one hole. After transplanting, place the seedlings in the greenhouse for a few days under shade, and then carry out normal cultivation. The survival rate of hardening and transplanting is more than 90%.

In a second aspect, the present invention provides a method for planting nasturtium, which comprises the aseptic rapid propagation method of nasturtium of any of the aforementioned embodiments.

The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

Example 1

This embodiment provides a method for aseptic rapid propagation of Trollius, which comprises the following steps:

S1. Obtain sterile seeds.

Wrap the nasturtium seeds in sterilized gauze, soak and clean them in detergent for 20 minutes, and then rinse them with clean water for 30 minutes. Move the cleaned nasturtium seeds to a clean bench, disinfect them with 75% alcohol for 1 minute, and then disinfect them with 5% sodium hypochlorite for 30 minutes to obtain sterile seeds.

S2. Inoculate the sterile seeds into a germination medium for germination to obtain nasturtium germination seedlings (see Figure 1).

First, based on MS medium, the sucrose in the MS medium was replaced with 25 mg/L white sugar, and the amount of agar was reduced to 6 g/L to obtain a basal medium, the basal medium was sterilized at high temperature, and then 25 mg/L of cephalosporin and 30 mg/L of a 1:1 mixture of 70% mancozeb and 75% thiophanate-methyl were added to the basal medium for disinfection. After the disinfection was completed, 50 mg/L activated carbon and 100 mg/L vitamin C were added to the basal medium, and then 2 mg/L 6-BA and 0.1 mg/L NAA were added to obtain a germination medium.

S3. Artificially cultivate the nasturtium germination seedlings to obtain tissue culture seedlings.

(1) Transplant the nasturtium seedlings into a sterilized optimal germination medium, inoculate 6-8 nasturtium seedlings per bottle, and culture them artificially in an artificial culture room at a temperature of 25±2°C, a humidity of 70%, and a photoperiod of 12 h light/12 h dark to obtain nasturtium sterile seedlings (see Figure 2).

(2) On sterile filter paper, leaves were removed with a scalpel and cut into stem segments containing axillary bud nodes. The stem segments of the sterile trollius seedlings were used as explants, and the explants were inserted into a proliferation and subculture medium in an artificial culture room under the following culture conditions: temperature 25±2°C, humidity 70%, and photoperiod 12 h light/12 h dark, to obtain clustered bud stem segments (see FIG3 ); wherein the proliferation and subculture medium was obtained by adding 2 mg/L 6-BA and 0.3 mg/L NAA to MS medium, and then adding 50 mg/L activated carbon and 100 mg/L vitamin C.

(3) The clustered bud stem segments were inoculated into a rooting medium to obtain tissue culture seedlings (see FIG. 4 ), wherein the rooting medium was obtained by adding 0.5 mg/L NAA, 50 mg/L activated carbon and 100 mg/L vitamin C to 1/2 MS medium.

S4. Harden and transplant the tissue culture seedlings.

Before transplanting the golden lotus tissue culture seedlings, hardening the seedlings can improve the resistance of the tissue culture seedlings to adverse environments and improve the survival rate of the seedlings after transplanting. After the golden lotus tissue culture seedlings take root for 30 days, transfer the tissue culture seedlings to the greenhouse for natural light exposure (avoid direct sunlight). The indoor temperature should be close to the temperature of the culture room (23℃-25℃), so that the seedlings can slowly adapt to the external environmental conditions to improve the adaptability of the golden lotus tissue culture seedlings to the external environment. Half open the bottle cap after 2 days, add a little sterile water to cover the surface of the culture medium, leave it for two days, completely open the bottle cap after 3 days, and transplant it after 3d-5d. Then take out the golden lotus tissue culture seedlings from the tissue culture bottle, clean the culture medium on the roots with water to prevent bacteria and other diseases. Do not hurt the root system when cleaning, then soak it in 1% carbendazim for 10 minutes, and then plant it in a plug tray. Make a small hole in the middle of the hole in the plug tray, insert the root system, cover the root system with a mixture of peat soil and perlite in a ratio of 3:1 (sterilized at high temperature in a sterilizer), and water thoroughly. When transplanting, plant one seedling in one hole. After transplanting, place the seedlings in the greenhouse for a few days under shade cultivation, and then carry out normal cultivation. The survival rate of hardening and transplanting reaches more than 90%.

Example 2

This embodiment provides a method for aseptic rapid propagation of Trollius, which comprises the following steps:

S1. Obtain sterile seeds.

Wrap the nasturtium seeds in sterilized gauze, soak and clean them in detergent for 15 minutes, and then rinse them with clean water for 35 minutes. Move the cleaned nasturtium seeds to a clean bench, disinfect them with 75% alcohol for 2 minutes, and then disinfect them with 5% sodium hypochlorite for 20 minutes to obtain sterile seeds.

S2. Inoculate the sterile seeds into a germination medium for germination to obtain nasturtium seedlings.

First, based on MS medium, the sucrose in the MS medium was replaced with 23 mg/L white sugar, and the amount of agar was reduced to 5 g/L to obtain a basal medium, the basal medium was sterilized at high temperature, and then 25 mg/L of cephalosporin and 20 mg/L of a 1:1 mixture of 75% mancozeb and 80% thiophanate-methyl were added to the basal medium for disinfection. After the disinfection was completed, 40 mg/L activated carbon and 120 mg/L vitamin C were added to the basal medium, and then 1.5 mg/L 6-BA and 0.1 mg/L NAA were added to obtain a germination medium.

S3. Artificially cultivate the nasturtium germination seedlings to obtain tissue culture seedlings.

(1) Transplanting nasturtium seedlings into a sterilized optimal germination medium, inoculating 6 to 8 nasturtium seedlings into each bottle, and artificially culturing them in an artificial culture room with a temperature of 25±2° C., a humidity of 70%, and a photoperiod of 12 h light/12 h dark, to obtain nasturtium sterile seedlings.

(2) On sterile filter paper, leaves are removed with a scalpel and cut into stem segments containing axillary bud nodes. The stem segments of the sterile nasturtium seedlings are used as explants, and the explants are inserted into a proliferation and subculture medium in an artificial culture room under the following culture conditions: temperature 25±2°C, humidity 70%, and photoperiod 12 h light/12 h dark, to obtain clustered bud stem segments; wherein the proliferation and subculture medium is obtained by adding 1.5 mg/L 6-BA and 0.3 mg/L NAA, and then adding 40 mg/L activated carbon and 120 mg/L vitamin C to MS medium.

(3) The clustered bud stem segments are inoculated into a rooting medium to obtain tissue culture plantlets, wherein the rooting medium is obtained by adding 0.4 mg/L NAA, 40 mg/L activated carbon and 120 mg/L vitamin C to 1/2 MS medium.

S4. Adopt the same method as Example 1 to harden and transplant the tissue culture seedlings.

Example 3

This embodiment provides a method for aseptic rapid propagation of Trollius, which comprises the following steps:

S1. Obtain sterile seeds.

Wrap the nasturtium seeds in sterilized gauze, soak and clean them in detergent for 25 minutes, and then rinse them with clean water for 25 minutes. Move the cleaned nasturtium seeds to a clean bench, disinfect them with 75% alcohol for 1 minute, and then disinfect them with 6% sodium hypochlorite for 25 minutes to obtain sterile seeds.

S2. Inoculate the sterile seeds into a germination medium for germination to obtain nasturtium germination seedlings.

First, based on MS medium, the sucrose in the MS medium was replaced with 27 mg/L white sugar, and the amount of agar was reduced to 6 g/L to obtain a basal medium, the basal medium was sterilized at high temperature, and then 25 mg/L of cephalosporin and 40 mg/L of a 1:1 mixture of 65% mancozeb and 70% thiophanate-methyl were added to the basal medium for disinfection. After the disinfection was completed, 60 mg/L activated carbon and 80 mg/L vitamin C were added to the basal medium, and then 2.5 mg/L 6-BA and 0.2 mg/L NAA were added to obtain a germination medium.

S3. Artificially cultivate the nasturtium germination seedlings to obtain tissue culture seedlings.

(1) Transplanting nasturtium seedlings into a sterilized optimal germination medium, inoculating 6 to 8 nasturtium seedlings per bottle, and artificially culturing them in an artificial culture room with a temperature of 25±2° C., a humidity of 70%, and a photoperiod of 12 h light/12 h dark, to obtain nasturtium sterile seedlings.

(2) On sterile filter paper, leaves are removed with a scalpel and cut into stem segments containing axillary bud nodes. The stem segments of sterile trollius seedlings are used as explants, and the explants are inserted into a proliferation and subculture medium in an artificial culture room under the following culture conditions: temperature 25±2°C, humidity 70%, and photoperiod 12 h light/12 h dark, to obtain clustered bud stem segments; wherein the clustered bud medium is obtained by adding 2.5 mg/L 6-BA and 0.4 mg/L NAA, and then adding 60 mg/L activated carbon and 80 mg/L vitamin C to MS medium.

(3) The clustered bud stem segments are inoculated into a rooting medium to obtain tissue culture plantlets, wherein the rooting medium is obtained by adding 0.6 mg/L NAA, 60 mg/L activated carbon and 80 mg/L vitamin C to 1/2 MS medium.

S4. Adopt the same method as Example 1 to harden and transplant the tissue culture seedlings.

Comparative Example 1

In this comparative example, the step S1 in Example 1 of "disinfection with 75% alcohol for 1 min, and then disinfection with 5% sodium hypochlorite for 30 min" is replaced by "sterilization with 0.1% mercuric chloride".

Since mercuric chloride is a dangerous substance, the results of this comparative example were obtained by referring to the literature: although the sterilization effect achieved by using 0.1% mercuric chloride for sterilization is better than that achieved by using 5% sodium hypochlorite for disinfection for 30 minutes, mercuric chloride is highly toxic and can pollute the body and the environment. Moreover, when 0.1% mercuric chloride is used to sterilize nasturtium seeds, although the contamination rate is reduced, the germination rate and survival rate of the nasturtium seeds are reduced. When 0.1% mercuric chloride is used to sterilize nasturtium seeds for 20 minutes, the contamination rate of the nasturtium is 6.05%, but the germination rate of its seeds is only 22.55%.

Comparative Examples 2-4

In this comparative example, the step S1 of Example 1, "disinfect with 75% alcohol for 1 min, and then disinfect with 5% sodium hypochlorite for 30 min" is modified to "disinfect with 75% alcohol for 1 min, and then disinfect with 2% sodium hypochlorite for 10, 20 and 30 min".

Among them, the contamination rate of the nasturtium seeds obtained in comparative example 2 was 62.22%, and the germination rate was 34.45%. The contamination rate of the nasturtium seeds obtained in comparative example 3 was 53.89%, and the germination rate was 46.12%. The contamination rate of the nasturtium seeds obtained in comparative example 4 was 47.78%, and the germination rate was 58.89%.

Comparative Example 5

In this comparative example, the step S1 in Example 1, "disinfect with 75% alcohol for 1 minute, and then disinfect with 5% sodium hypochlorite for 30 minutes", was modified to "disinfect with 75% alcohol for 1 minute, and then disinfect with 5% sodium hypochlorite for 10 minutes". The contamination rate of the obtained nasturtium seeds was 35.56%, and the germination rate was 38.89%.

Comparative Examples 6-8

In this comparative example, the step S1 of Example 1, "disinfect with 75% alcohol for 1 min, and then disinfect with 5% sodium hypochlorite for 30 min" is modified to "disinfect with 75% alcohol for 1 min, and then disinfect with 10% sodium hypochlorite for 10, 20 and 30 min".

Among them, the contamination rate of the nasturtium seeds obtained in Comparative Example 6 was 12.78%, and the germination rate was 38.89%. The contamination rate of the nasturtium seeds obtained in Comparative Example 7 was 1.11%, and the germination rate was 28.89%. The contamination rate of the nasturtium seeds obtained in Comparative Example 8 was 0%, achieving a good sterilization effect, but the germination rate of the nasturtium seeds was greatly reduced, and the germination rate was 13.89%.

Comparative Example 9

In this comparative example, the step S1 in Example 1 of "disinfecting with 75% alcohol for 1 min, and then disinfecting with 5% sodium hypochlorite for 30 min" is modified to "disinfecting with 5% sodium hypochlorite for 30 min".

When disinfected with 5% sodium hypochlorite for 30 minutes, the contamination rate of the nasturtium seeds was 24.67% and the germination rate was 60.67%.

Comparative Example 10

In this comparative example, step S1 of Example 1, "adding 25 mg/L of cephalosporin and 30 mg/L of a 1:1 mixture of 70% mancozeb and 75% thiophanate-methyl to the basal culture medium for disinfection" was omitted, and only high temperature sterilization was used.

When the basic culture medium is only sterilized by high temperature, contamination phenomena such as slimy substances appearing around the culture materials, moldy growth in some parts of the culture medium, and the production of villous hyphae and pathogenic spores may occur during the tissue culture of the nasturtium.

Comparative Example 11

In this comparative example, the step S1 of Example 1, "adding 25 mg/L of cephalosporin and 30 mg/L of a 1:1 mixture of 70% mancozeb and 75% thiophanate-methyl to the basal culture medium for disinfection" was modified to "adding 25 mg/L of cephalosporin to the basal culture medium for disinfection".

During the tissue culture process of Nasturtium, some parts of the culture medium may become moldy, producing villous hyphae and pathogenic spores and other contamination phenomena.

Comparative Example 12

In this comparative example, the step S1 in Example 1 of "adding 25 mg/L of cephalosporin and 30 mg/L of a 1:1 mixture of 70% mancozeb and 75% thiophanate-methyl to the basal culture medium for disinfection" was modified to "adding 30 mg/L of a 1:1 mixture of 70% mancozeb and 75% thiophanate-methyl to the basal culture medium for disinfection".

During the nasturtium tissue culture process, the culture medium becomes turbid and water-soaked, and sometimes even a mucus-like substance appears around the entire culture material.

Comparative Example 13

In this comparative example, the step S1 of Example 1 "adding 50 mg/L activated carbon and 100 mg/L vitamin C" was deleted.

In the early stage, the stem segments of the golden lotus turned heavily brown, the clustered buds grew weakly, and in the later stage, all of them turned brown.

Comparative Example 14

In this comparative example, the step S1 in Example 1, "adding 50 mg/L activated carbon and 100 mg/L vitamin C" was replaced by "adding 50 mg/L activated carbon".

The leaves of the nasturtium turn yellow, the seedlings grow weakly, the browning rate reaches 65%, and the degree of browning is severe.

Comparative Example 15

In this comparative example, the “addition of 40-60 mg/L activated carbon and 80-120 mg/L vitamin C” in step S1 of Example 1 was replaced by “100 mg/L vitamin C”.

The golden lotus seedlings grew well, with a browning rate of 30% and a medium degree of browning.

Comparative Example 16

In this comparative example, the "germination medium 2 mg/L 6-BA and 0.1 mg/L NAA" in step S1 of Example 1 was replaced by "0.1 mg/LTDZ".

The germination rate of the obtained nasturtium seeds was 44.67%, and the leaves of the germinated seedlings were emerald green and the roots were thin and long.

Comparative Example 17

In this comparative example, the "germination medium 2 mg/L 6-BA and 0.1 mg/L NAA" in step S1 of Example 1 was replaced with "0.1 mg/L IBA".

The germination rate of the obtained nasturtium seeds was 30.67%, and the germinated seedlings had no wrinkled leaves and the leaves were light green.

Experimental Example 1: Experiment on the contamination rate and germination rate of nasturtium seeds.

The sterilization methods of Examples 1-2 and Comparative Examples 1-9 were subjected to contamination rate experiments. Please refer to Table 1 for the experimental results.

Table 1. Statistical table of the contamination rate experimental results of golden lotus seeds

deal with Approach Contamination rate (%) Germination rate (%) Example 1 75% ethanol for 1 min, 5% sodium hypochlorite for 30 min 10.55±2.56 ^f 76.67±2.89 ^a Example 2 75% ethanol for 1 min, 5% sodium hypochlorite for 20 min 20.00±3.34 ^e 57.23±0.96 ^b Comparative Example 1 0.1% mercuric chloride 20min 6.05 22.55 Comparative Example 2 75% ethanol for 1 min, 2% sodium hypochlorite for 10 min 62.22±3.85 ^a 34.45±4.19 ^de Comparative Example 3 75% ethanol for 1 min, 2% sodium hypochlorite for 20 min 53.89±4.20 ^b 46.12±4.19 ^c Comparative Example 4 75% ethanol for 1 min, 2% sodium hypochlorite for 30 min 47.78±1.92 ^c 58.89±2.55 ^b Comparative Example 5 75% ethanol for 1 min, 5% sodium hypochlorite for 10 min 35.56±3.85 ^d 38.89±1.92 ^d Comparative Example 6 75% ethanol for 1 min, 10% sodium hypochlorite for 10 min 12.78±1.92 ^f 38.89±4.20 ^d Comparative Example 7 75% ethanol for 1 min, 10% sodium hypochlorite for 20 min 1.11±1.93 ^g 28.89±2.55 ^e Comparative Example 8 75% ethanol for 1 min, 10% sodium hypochlorite for 30 min 0 ^g 13.89±0.96 ^f Comparative Example 9 5% sodium hypochlorite disinfection for 30 minutes 24.67 60.67

It should be noted that Table 1 mainly performs one-way ANOVA data processing on the ethanol + sodium hypochlorite treatment method of Examples 1-2 and Comparative Examples 2-8, while the data of Comparative Examples 1 and 9 are not processed by one-way ANOVA data processing, so only the corresponding contamination rate and germination rate are listed, which does not affect the basic judgment of all data.

Furthermore, the optimal germination medium was screened by changing the amount of 6-BA and NAA added to different germination mediums. The screening results are shown in Table 2.

Table 2. Germination rate experiment of nasturtium seeds

Based on the above screening, in the present application, the amount of 6-BA and NAA added to the germination medium is preferably 2 mg/L 6-BA and 0.1 mg/L NAA.

Experimental Example 2: Bacterial infection experiment of nasturtium tissue culture.

Due to the endophytic bacteria carried by nasturtium itself and the human and environmental pollution caused by incomplete sterilization, nasturtium will be contaminated with bacteria during tissue culture. Common bacteria include bacteria in the form of turbid water stains and mucus and fungi in the form of villi that produce a variety of pathogenic spores. Adding cephalosporins, an antibiotic that has a good effect on the culture medium to resist bacteria, and a mixture of mancozeb and thiophanate-methyl, which have a strong contact fungicidal effect, can significantly reduce the risk of tissue culture materials being contaminated with bacteria, and has little effect on the growth and development of nasturtium.

Experimental Example 3: Browning experiment of seedlings during cultivation.

The optimal browning conditions were obtained by changing the dosage of added vitamin C and activated carbon. Please refer to Table 3 for the operating conditions and results.

Table 3. Screening of optimal browning conditions

It can be seen from the above table that the combination of activated carbon and vitamin C used in this application can achieve unexpected technical effects.

Experimental Example 4: Proliferation rate experiment.

Table 4. Statistical table of proliferation rate experimental results

As can be seen from Table 4, the preferred components of the proliferation and subculture medium in the present application include: MS medium obtained by adding 2 mg/L 6-BA and 0.3-0.5 mg/L NAA.

Experimental Example 5: Rooting rate and transplanting survival rate experiment.

Table 5. Statistical table of rooting rate experiment of golden lotus

Auxin type and concentration Rooting rate/% Average number of roots/line Root Characteristics 0.1mg/LNAA 60 4.5 Thick roots, white adventitious roots 0.5mg/L NAA 100 8.1 The root is thick and has yellow hairs 0.1mg/LIBA 66.67 4.67 The root is slender and has yellow hairs 0.5mg/LIBA 86.67 6.8 The root is slender and has yellow hairs

As can be seen from the above table, the effect obtained by adding the LNAA auxin in this example is significantly better than that of other auxins.

Experimental Example 6: Nasturtium transplant survival rate experiment

Table 6. Statistics of transfer survival rate

Matrix solution Survival rate (%) Peat soil 75 Peat soil: perlite = 1:1 85 Peat soil: perlite = 3:1 90

It can be seen from the above table that the substrate solution of peat soil: perlite = 3:1 used in this application can obtain a better survival rate.

In summary, the aseptic rapid propagation method of nasturtium provided in the present application uses 75% alcohol + 4-6% sodium hypochlorite to disinfect nasturtium seeds, which has good disinfection effect and will not have adverse effects on explant activity and the environment. Further, in the present application, 20-30mg/L of cephalosporin and 20-40mg/L 65-75% mancozeb and 70-80% thiophanate-methyl 1:1 mixed solution is added to the basal culture medium for disinfection, which can further reduce the contamination rate of seed germination and will not affect the survival rate of seedlings. And in subsequent experiments, there will be no re-infection phenomenon due to the production of endophytes by nasturtium itself or incomplete disinfection. The addition of activated carbon and vitamin C can play a synergistic role, which can not only effectively improve browning, but also avoid affecting the growth and differentiation of seedlings. In the present application, the above-mentioned treatment can effectively improve the poor disinfection effect of nasturtium, the serious problems of bacterial infection and browning imagination, which is conducive to improving the seedling rate of nasturtium tissue culture.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (3)

1. A method for aseptic rapid propagation of Trollius, characterized in that it comprises: The cleaned nasturtium seeds are sterilized with 75% alcohol for 1-2 minutes, and then sterilized with 4-6% sodium hypochlorite for 20-30 minutes to obtain sterile seeds; The sterile seeds are inoculated into a germination medium for germination to obtain nasturtium germination seedlings; wherein the germination medium is obtained by first adding 20-30 mg/L of cephalosporin and 20-40 mg/L of a mixed solution to a basic medium, and then adding 40-60 mg/L of activated carbon and 80-120 mg/L of vitamin C, wherein the mixed solution is 65-75% mancozeb and 70-80% thiophanate-methyl mixed in a ratio of 1:1; the basic medium is based on MS medium + 2 mg/L 6-BA + 0.1 mg/L NAA, sucrose in the MS medium is replaced by 23-27 mg/L of white sugar, and the amount of agar is reduced to 5-6 g/L; Artificially cultivating the nasturtium germination seedlings to obtain tissue culture seedlings, and hardening and transplanting the tissue culture seedlings; hardening and transplanting the tissue culture seedlings include: placing the tissue culture seedlings in the tissue culture bottle in a greenhouse for hardening 5 days in advance, the substrate is a mixture of peat soil and perlite, the volume ratio of peat soil to perlite in the mixture is 3:1, high-temperature sterilization is performed in a sterilizing pot, the pH value is adjusted to 5.8, and then the tissue culture seedlings are taken out from the tissue culture bottle, the culture medium on the roots is cleaned with water, soaked in 1% carbendazim for 10 minutes, and then planted in a plug tray; Artificially cultivating the nasturtium germination seedlings to obtain tissue culture seedlings comprises: Transplanting the nasturtium germination seedlings into a sterilized germination medium for further artificial germination culture to obtain nasturtium sterile seedlings, wherein the artificial germination culture is first dark culture for seven days, followed by 12 hours of light/12 hours of darkness, with a light intensity of 1300-1700 Lux, and a germination culture time of 28-30 days; The stem segments of the aseptic seedlings of the golden lotus are used as explants, and the explants are inserted into a proliferation and subculture medium for proliferation and subculture, wherein the components of the proliferation and subculture medium are MS medium + 2mg/L 6-BA + 0.3-0.5mg/L NAA + 40-60mg/L activated carbon + 80-120mg/L vitamin C, and the time of the proliferation and subculture is 58-62d; clustered bud stem segments are obtained; and the clustered bud stem segments are inoculated into a rooting medium for culture, wherein the rooting medium is 1/2MS medium + 0.5mg/L NAA + 40-60mg/L activated carbon + 80-120mg/L vitamin C, and the time of the rooting culture is 28-32d, so as to obtain tissue culture seedlings. 2. The aseptic rapid propagation method of nasturtium according to claim 1 is characterized in that cleaning the nasturtium seeds comprises: wrapping the nasturtium seeds in sterilized gauze, soaking and cleaning them in detergent for 15-25 minutes, and then rinsing them with clean water for 25-35 minutes. 3. A method for planting nasturtium, characterized in that it comprises the aseptic rapid propagation method of nasturtium according to any one of claims 1-2.