CN109452330B - A kind of bacteriostatic agent for plant tissue culture and its application in the tissue culture of clematis - Google Patents

Abstract

The invention discloses a bacteriostatic agent for plant tissue culture and its application in the tissue culture of clematis. 500ml; 5-20g of chitosan oligosaccharide; 5-20ml of surfactant; the rest is water; the plant extract is the ethanol extract of the mixture of garlic bulb, ginger rhizome, bitter gourd peel and pomegranate peel. The invention uses garlic bulbs, ginger rhizomes, bitter gourd peels and pomegranate peels as raw materials to prepare plant extracts, and combines the plant extracts with chitosan oligosaccharides to obtain bacteriostatic agents, which can effectively reduce bacteria and bacteria in tissue culture. Fungal contamination rate, and significantly improve the seedling rate of clematis tissue culture, reduce production costs, and can be widely used in plant biotechnology fields such as tissue culture rapid propagation, seedling detoxification and regeneration plant line establishment.

Description

A kind of bacteriostatic agent for plant tissue culture and its application in the tissue culture of clematis

technical field

The invention relates to the technical field of plant tissue culture, in particular to a bacteriostatic agent for plant tissue culture and its application in the tissue culture of Clematis.

Background technique

Clematis is a traditional precious medicinal material in my country. (Anoectochilus) perennial herb. "Fujian Medicine Records" records that its properties are sweet and flat, clearing heat and cooling blood, expelling wind and dampness, and treating hemoptysis, bronchitis, tuberculous meningitis, nephritis, cystitis, urinary tract stones, rheumatoid arthritis, and minor acute Convulsions, Pediatric Tetanus. "The Sea of Chinese Medicine" states that its properties are sweet and savory, enter the lung, liver, kidney, and bladder meridians. Unfavorable and dripping astringent pain and so on. Modern medicine shows that clematis mainly contains flavonoids, sugars, volatile oils, alkaloids, organic acids, amino acids, steroids, etc. The pharmacological effects of clematis and its extracts are mainly manifested as hypoglycemic, hypotensive, antiviral, antioxidant and other pharmacological effects are still being discovered. With in-depth research, the excellent quality of clematis has become increasingly prominent, favored by the world, and has broad prospects for development and utilization. Clematis is distributed in Guangxi, Guangdong, Hainan, Guizhou, Sichuan, Yunnan and other provinces in my country; but in recent years, people's long-term mining of clematis has led to scarcity of its germplasm resources. At present, the large-scale production of clematis can be achieved by using plant tissue culture technology to meet people's medicinal, research and ornamental needs.

The large-scale production of clematis tissue culture (hereinafter referred to as "tissue culture") has developed rapidly in my country, and tissue culture technology has been gradually improved, but it often presents different degrees of pollution in tissue culture. At the stage of primary culture, due to pollution problems, the cost of obtaining sterile seedlings is high, or although sterile seedlings are obtained in primary culture, a large amount of pollution occurs during subculture, which keeps the cost of tissue culture high.

Therefore, reducing the contamination rate of tissue culture is an important technical link in large-scale production, and it is also one of the important indicators to measure the cost, risk and feasibility of tissue culture work. From the perspective of the whole process of tissue culture, different degrees of pollution may occur in each link. In order to reduce the pollution rate, in the process of tissue culture, in addition to formulating a standardized process of aseptic operation, focusing on aseptic operation awareness, training and supervision, At the same time, adding broad-spectrum bacteriostatic agents to the medium is also an important method to reduce bacterial and fungal contamination. The research, screening and promotion of effective bacteriostatic agents can reduce fungal and bacterial contamination, simplify aseptic operations, and reduce the cost and risk of tissue culture.

SUMMARY OF THE INVENTION

The invention provides a bacteriostatic agent for plant tissue culture and its application in clematis The seedling rate of lotus tissue culture reduces production costs, and can be widely used in plant biotechnology fields such as tissue culture rapid propagation, seedling detoxification, and establishment of regenerated plant lines.

The technical scheme provided by the present invention is specifically as follows:

A bacteriostatic agent for plant tissue culture, calculated in 1L, comprising the following components:

Plant extract 100～500ml;

Chitooligosaccharide 5～20g;

Surfactant 5～20ml;

The rest is water;

The plant extract is an ethanol extract of a mixture of garlic bulb, ginger rhizome, bitter gourd peel and pomegranate peel.

Preferably, the bacteriostatic agent for plant tissue culture, calculated in 1L, includes the following components:

Plant extract 200ml;

Chitooligosaccharide 20g;

Surfactant 10ml;

The rest is water;

The plant extract is an ethanol extract of a mixture of garlic bulb, ginger rhizome, bitter gourd peel and pomegranate peel.

More preferably, the surfactant is tween 20.

It has been found through experiments that the combination of the above-mentioned plant extract and chitosan oligosaccharide can effectively inhibit bacteria and fungi produced in the process of tissue culture, especially Bacillus, Pseudomonas, Streptomyces, Rhododendron and Penicillium.

Specifically, the preparation method of the plant extract comprises:

(1) after garlic bulb, ginger rhizome, balsam pear peel and pomegranate peel are mixed, add in ethanol, stir and pulverize to obtain mixed solution;

(2) After the mixed solution is subjected to ultrasonic treatment, solid impurities are removed by filtration, and then ethanol is removed by evaporation and concentration to obtain a plant extract.

Preferably, in step (1), the mass ratio of the garlic bulb, ginger rhizome, bitter gourd peel and pomegranate peel is 1-3:1-2:1-2:1-2.

Preferably, in step (2), the temperature of the ultrasonic treatment is 40-70° C., and the frequency is 30-50 KHz.

The present invention also provides a method for clematis tissue culture using the bacteriostatic agent for plant tissue culture, comprising:

(1) preparation of explant: take the explant of Clematis clematis, place it in the bacteriostatic agent for plant tissue culture after cleaning, soak and disinfect, take out and rinse, obtain sterilized explant;

(2) Induction culture: the sterilized explants are inoculated into the induction medium containing the bacteriostatic agent for plant tissue culture, and the induction culture is carried out to obtain callus containing clustered buds;

(3) proliferation subculture: inoculate the callus containing the clump bud in the proliferation subculture medium containing the bacteriostatic agent for plant tissue culture, carry out the proliferation subculture, and obtain the clump seedling;

(4) rooting culture: inoculate the clump seedlings in the rooting medium containing the bacteriostatic agent for the plant tissue culture, carry out rooting culture, and obtain tissue culture seedlings;

(5) Refining and transplanting.

The explants are bulbs, seeds or leaves of clematis.

Preferably, the induction medium is MS+sucrose 20-30g·L ^-1 + agar 5-10g·L ^-1 +6-BA 1.0-2.0mg·L ^-1 +NAA 0.2-1.0mg·L ^-1 + GA 1.5mg·L ^-1 + bacteriostatic agent 200-300ml·L ^-1 .

Preferably, the proliferation subculture medium is MS+sucrose 20-30g·L ^-1 + agar 5-10g·L ^-1 +6-BA 0.5-1.5mg·L ^-1 +NAA 0.2-0.5mg·L ^-1 + bacteriostatic agent 300-400ml·L ^-1 .

Preferably, the rooting medium is 1/2MS+sucrose 20-30g·L ^-1 +agar 5-10g·L ^-1 +NAA 0.3-0.5mg·L ^-1 +bacteriostatic agent 100-150ml·L ^-1 .

Compared with the prior art, the present invention has the following beneficial effects:

The invention uses garlic bulbs, ginger rhizomes, bitter gourd peels and pomegranate peels as raw materials to prepare plant extracts, and combines the plant extracts with chitosan oligosaccharides to obtain bacteriostatic agents, which can effectively reduce bacteria and bacteria in tissue culture. It can be widely used in the field of plant biotechnology such as tissue culture rapid propagation, seedling detoxification and regeneration plant line establishment.

Description of drawings

Fig. 1 is the bacteriostatic effect of plant tissue culture bacteriostatic agent to different bacteria in embodiment 2;

Among them, A is Pseudomonas; B is Bacillus; C is Enterobacter aquaticus.

Fig. 2 is the bacteriostatic effect of plant tissue culture bacteriostatic agent to different fungi in Example 2;

Among them, A1 is the experimental group _of Alternaria fungi _; A2 is the control group of Alternaria fungi; B is Rhodotorula rubra; _C1 is the experimental group of Penicillium; _C2 is the control group of Penicillium.

Fig. 3 is the bacteriostatic effect test of plant tissue culture bacteriostatic agent to Penicillium in Example 3;

Among them, A and B are inoculated with penicillin; C and D are medium containing bacteriostatic agent + inoculated with penicillin; E and F are without penicillin inoculation.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited to the given embodiments.

Example 1 Preparation of bacteriostatic agent for plant tissue culture

1. Preparation of plant extract:

(1) get 40g garlic bulb, 20g ginger rhizome, 20g bitter gourd peel and 20g pomegranate peel, after cleaning, add in the 95% ethanol of 100ml, stir and pulverize to obtain mixed solution;

(2) above-mentioned mixed solution is carried out ultrasonic treatment, the temperature of ultrasonic treatment is 60 ℃, frequency is 40KHz, time is 30min, after filtering and removing solid impurity, then adopt rotary evaporator to remove ethanol to obtain concentrated solution, adopt pure water to concentrate solution Dilute the volume to 100ml to obtain a plant extract.

2. Preparation of bacteriostatic agent for plant tissue culture:

Take 20ml of plant extract, 2g of chitosan oligosaccharide, 1ml of co-solvent tween20 and 60ml of pure water, put it into a 100ml volumetric flask, mix well, dilute to 100ml with deionized water, pour it into a reagent bottle, and place it in a 4°C refrigerator Save it as a backup.

Comparative Example 1

1. Preparation of plant extract:

(1) get 60g garlic bulbs and 40g ginger rhizomes, after cleaning, add in the 95% ethanol of 100ml, stir and pulverize to obtain mixed solution;

(2) above-mentioned mixed solution is carried out ultrasonic treatment, the temperature of ultrasonic treatment is 60 ℃, frequency is 40KHz, time is 30min, after filtering and removing solid impurity, then adopt rotary evaporator to remove ethanol to obtain concentrated solution, adopt pure water to concentrate solution Dilute the volume to 100ml to obtain a plant extract.

2. Preparation of bacteriostatic agent for plant tissue culture:

Take 20ml of plant extract, 2g of chitosan oligosaccharide, 1ml of co-solvent tween20 and 60ml of pure water, put it into a 100ml volumetric flask, mix well, dilute to 100ml with deionized water, pour it into a reagent bottle, and place it in a 4°C refrigerator Save in spare.

Comparative Example 2

1. Preparation of plant extract:

(1) get 40g garlic bulb, 20g ginger rhizome, 40g bitter gourd peel, after cleaning, add in the 95% ethanol of 100ml, stir and pulverize to obtain mixed solution;

(2) above-mentioned mixed solution is carried out ultrasonic treatment, the temperature of ultrasonic treatment is 60 ℃, frequency is 40KHz, time is 30min, after filtering and removing solid impurity, then adopt rotary evaporator to remove ethanol to obtain concentrated solution, adopt pure water to concentrate solution Dilute the volume to 100ml to obtain a plant extract.

2. Preparation of bacteriostatic agent for plant tissue culture:

Take 20ml of plant extract, 2g of chitosan oligosaccharide, 1ml of co-solvent tween20 and 60ml of pure water, put it into a 100ml volumetric flask, mix well, dilute to 100ml with deionized water, pour it into a reagent bottle, and place it in a 4°C refrigerator Save in spare.

Comparative Example 3

1. Preparation of plant extract:

(1) get 40g garlic bulb, 20g ginger rhizome, 40g pomegranate peel, after cleaning, add in the 95% ethanol of 100ml, stir and pulverize to obtain mixed solution;

(2) above-mentioned mixed solution is carried out ultrasonic treatment, the temperature of ultrasonic treatment is 60 ℃, frequency is 40KHz, time is 30min, after filtering and removing solid impurity, then adopt rotary evaporator to remove ethanol to obtain concentrated solution, adopt pure water to concentrate solution Dilute the volume to 100ml to obtain a plant extract.

2. Preparation of bacteriostatic agent for plant tissue culture:

Take 20ml of plant extract, 2g of chitosan oligosaccharide, 1ml of co-solvent tween20 and 60ml of pure water, put it into a 100ml volumetric flask, mix well, dilute to 100ml with deionized water, pour it into a reagent bottle, and place it in a 4°C refrigerator Save in spare.

Comparative Example 4

1. Preparation of plant extract:

(1) get 40g garlic bulb, 20g ginger rhizome, 20g bitter gourd peel and 20g pomegranate peel, after cleaning, add in the 95% ethanol of 100ml, stir and pulverize to obtain mixed solution;

(2) above-mentioned mixed solution is carried out ultrasonic treatment, the temperature of ultrasonic treatment is 60 ℃, frequency is 40KHz, time is 30min, after filtering and removing solid impurity, then adopt rotary evaporator to remove ethanol to obtain concentrated solution, adopt pure water to concentrate solution Dilute the volume to 100ml to obtain a plant extract.

2. Preparation of bacteriostatic agent for plant tissue culture:

Take 20ml of plant extract, 2g of chitosan, 1ml of co-solvent tween20 and 60ml of pure water, put it into a 100ml volumetric flask, mix well, dilute to 100ml with deionized water, pour it into a reagent bottle, and place it in a 4°C refrigerator Save it as a backup.

Example 2 Bacteriostatic activity of bacteriostatic agents for plant tissue culture against bacteria and fungi

The bacteriostatic agents for plant tissue culture prepared in Example 1 and Comparative Examples 1 to 4 were used to treat 3 strains of bacteria (Pseudomonas, Bacillus and Enterobacter hydrophila) and 3 strains of fungi (Alternaria alternata) in tissue culture contamination , Rhododendron, and Penicillium) to study the antibacterial effect.

The details are as follows:

Bacteriostasis test: Take an Oxford cup with a size of Φ7.8mm×6mm×10mm (outer diameter×inner diameter×height), put it at 121°C for autoclaving, and then dry and cool it for later use. Bacillus, Pseudomonas and Enterobacter aquaticus were spread on the surface of LB plates (90 mm in diameter) in a clean bench, each plate containing 200 μl of test bacteria at a concentration of 10 ⁶ CFU/ml. Put the Oxford cup on the surface of each plate, draw 200 μl of the bacteriostatic agent for plant tissue culture prepared in Example 1 and drop it into the Oxford cup, and then place it at 30° C. for 48-72 hours, with 3 repetitions for each group; LB dishes without bacteriostatic agents were used as controls.

Fungal bacteriostatic test: Take LB plate containing 100 μl of the bacteriostatic agent for plant tissue culture prepared in Example 1, and inoculate the bacterial plaques of the fungi Streptomyces, Rhododendron and Penicillium on the above-mentioned ultra-clean workbench. The surface of the LB plate (diameter 60mm) was placed at 25°C for 4-5 days, and each group was repeated with 3 replicates; the LB plate with bacteriostatic agents but no fungus inoculated was used as a control.

The test results are as follows:

Figure 1 shows that there is an obvious bacteriostatic zone around the Oxford cup containing the bacteriostatic agent for plant tissue culture prepared in Example 1;

Figure 2 shows that the growth of fungi in the plate containing the bacteriostatic agent for plant tissue culture prepared in Example 1 was significantly inhibited; wherein, the diameter of the antibacterial zone of Rhodotorula rubrum reached 16 mm, which was highly sensitive to the bacteriostatic agent, and Streptomyces and The bacteriostatic rate of Penicillium reached 90% ((control plaque diameter-test plaque diameter)/control plaque diameter).

The specific antibacterial effect data are shown in Table 1.

Table 1 The results of the bacteriostatic treatment of different bacterial species with the bacteriostatic agents for plant tissue culture prepared in Example 1 and Comparative Examples 1 to 4

Note: "+" means: the diameter of the bacterial circle is 9-10mm, mildly sensitive; "++" means: the diameter of the bacterial circle is 10-15mm, moderately sensitive; "+++" means: the diameter of the bacterial circle is 15-20mm, the height Sensitive; "++++" means: bacterial circle diameter >20mm, extremely sensitive.

Example 3 Inhibitory effect of bacteriostatic agent for plant tissue culture on Penicillium

In the ultra-clean workbench, Penicillium is smeared on the surface of clematis seedlings (tissue cultured seedlings obtained by tissue culture), respectively inoculated in the medium containing or not containing the bacteriostatic agent for plant tissue culture prepared in Example 1 , placed at 25°C, light 12h/d, light intensity 2000lx for 3-5d.

Test results: As shown in Figure 3, the contamination degree of the tissue culture seedlings containing the bacteriostatic agent was significantly lighter.

Example 4

A method for tissue culture of clematis using a bacteriostatic agent for plant tissue culture, the specific contents are as follows:

(1) preparation of explant: choose the bulb of clematis as explant, and the explant is washed with water for 20 minutes, put into the bacteriostatic agent for plant tissue culture prepared in Example 1 and soaked for 20min, and then use no The bacteria water was rinsed twice, the water was filtered, and the explants after surface sterilization were cut with a scalpel soaked in the bacteriostatic agent to obtain the explants to be inoculated;

(2) Preparation of medium: According to the composition and dosage of MS basic medium, configure corresponding mother liquor, successively take the mother liquor of macroelements, the mother liquor of trace elements, the mother liquor of iron salt and the organic components, and after mixing, press the induction medium, proliferation and subculture Hormones and the bacteriostatic agent for plant tissue culture prepared in Example 1 were added according to the components of the base and rooting medium.

Use a pH meter to measure the pH of the medium, and adjust the pH to 5.8 with 1M NaOH solution or 1M hydrochloric acid; then heat to dissolve the agar, and finally add water to make up the volume; divide the prepared medium into culture bottles and seal them with bottle caps. Autoclave at 121°C, cool to about 60°C, add the bacteriostatic agent for plant tissue culture prepared in Example 1 under aseptic conditions, and store it in a clean area for later use after solidification.

(3) induction culture: the sterilized clematis explants are inoculated on the induction medium, placed in the tissue culture room for induction culture, after culturing 20d, callus is obtained, and the edge of the bulb grows cluster buds;

Among them, the induction medium is: sucrose 30g·L ^-1 + agar 5g·L ^-1 + MS+6-BA 2.0mg·L ^-1 +NAA 1.0mg·L ^-1 +GA 1.5mg·L ^-1 + pH Inoculum (Example 1) 200ml·L ^-1 , pH to 5.8, autoclaved at 121°C; conditions for induction culture: temperature at 25°C, light at 12h/d, and light intensity at 1500lx.

(4) proliferation subculture: cut the induced clump buds, inoculate on the proliferation subculture medium, carry out the proliferation subculture, and transfer once every 25 days to obtain the clump seedlings;

Wherein, the proliferation subculture medium is: sucrose 30g·L ^-1 + agar 5g·L ^-1 +MS+6-BA 1.5mg·L ^-1 +NAA0.5mg·L ^-1 +bacteriostatic agent (Example 1 ) 300ml·L ^-1 , pH to 5.8, autoclaved at 121°C; conditions for proliferation and subculture were: temperature at 25°C, light at 12h/d, and light intensity at 1500lx.

(5) rooting culture: when the clump seedlings grow to 2～4cm, the clump seedlings are cut into individual plants, inoculated on the rooting medium, and the rooting culture is carried out, and after culturing for 25 days, a complete tissue culture seedling is obtained;

Wherein, the rooting medium is: sucrose 30g·L ^-1 + agar 5g·L ^-1 +1/2MS+NAA 0.3mg·L ^-1 + bacteriostatic agent (Example 1) 100ml·L ^-1 , pH to 5.8 , 121 ℃ high pressure sterilization; the conditions of rooting culture are: the temperature is 25 ℃, the light is 12h/d, and the light intensity is 1500lx.

(6) seedling hardening and transplanting: take out the complete tissue culture seedling after rooting culture, wash the medium of root, transplant in the plug tray, cultivate to obtain clematis;

Among them, the substrates were commonly used humus, perlite and pine bark; the upper layer of the greenhouse was covered with a shading net, and the ambient temperature for transplanting was 25 °C. Finally, the transplanting survival rate reached 86%.

Example 5

A method for tissue culture of clematis using a bacteriostatic agent for plant tissue culture, the specific contents are as follows:

(1) preparation of explants: choose the leaves of clematis as explants, rinse the explants with water for 20 minutes, put them into the bacteriostatic agent for plant tissue culture prepared in Example 1, and soak for 20 minutes, and then use no The bacteria water was rinsed twice, the water was filtered, and the explants after surface sterilization were cut with a scalpel soaked in the bacteriostatic agent to obtain the explants to be inoculated;

(2) Preparation of medium: According to the composition and dosage of MS basic medium, configure corresponding mother liquor, successively take the mother liquor of macroelements, the mother liquor of trace elements, the mother liquor of iron salt and the organic components, and after mixing, press the induction medium, proliferation and subculture Hormones and the bacteriostatic agent for plant tissue culture prepared in Example 1 were added according to the components of the base and rooting medium.

Use a pH meter to measure the pH of the medium, and adjust the pH to 5.8 with 1M NaOH solution or 1M hydrochloric acid; then heat to dissolve the agar, and finally add water to make up the volume; divide the prepared medium into culture bottles and seal them with bottle caps. Autoclave at 121°C, cool to about 60°C, add the bacteriostatic agent for plant tissue culture prepared in Example 1 under aseptic conditions, and store it in a clean area for later use after solidification.

(3) induction culture: the sterilized clematis explants are inoculated on the induction medium, placed in the tissue culture room for induction culture, and after culturing for 20 days, callus is obtained, and the edge of the leaf grows cluster buds;

Among them, the induction medium is: sucrose 30g·L ^-1 + agar 5g·L ^-1 + MS+6-BA 2.0mg·L ^-1 +NAA1.0mg·L ^-1 +GA 1.5mg·L ^-1 + pH Inoculum (Example 1) 200ml·L ^-1 , pH to 5.8, autoclaved at 121°C; conditions for induction culture: temperature at 25°C, light at 12h/d, and light intensity at 1500lx.

(4) proliferation subculture: cut the induced clump buds, inoculate on the proliferation subculture medium, carry out the proliferation subculture, and transfer once every 25 days to obtain the clump seedlings;

Wherein, the proliferation subculture medium is: sucrose 30g·L ^-1 + agar 5g·L ^-1 +MS+6-BA 1.5mg·L ^-1 +NAA0.5mg·L ^-1 +bacteriostatic agent (Example 1 ) 300ml·L ^-1 , pH to 5.8, autoclaved at 121°C; conditions for proliferation and subculture were: temperature at 25°C, light at 12h/d, and light intensity at 1500lx.

(5) rooting culture: when the clump seedlings grow to 2～4cm, the clump seedlings are cut into individual plants, inoculated on the rooting medium, and the rooting culture is carried out, and after culturing for 25 days, a complete tissue culture seedling is obtained;

Wherein, the rooting medium is: sucrose 30g·L ^-1 + agar 5g·L ^-1 +1/2MS+NAA 0.3mg·L ^-1 + bacteriostatic agent (Example 1) 100ml·L ^-1 , pH to 5.8 , 121 ℃ high pressure sterilization; the conditions of rooting culture are: the temperature is 25 ℃, the light is 12h/d, and the light intensity is 1500lx.

(6) seedling hardening and transplanting: take out the complete tissue culture seedling after rooting culture, wash the medium of root, transplant in the plug tray, cultivate to obtain clematis;

Among them, the substrates were commonly used humus, perlite and pine bark; the upper layer of the greenhouse was covered with a shading net, and the ambient temperature for transplanting was 25 °C. Finally, the transplanting survival rate reached 83.5%.

Comparative Example 5

A method for tissue culture of Clematis, the specific contents are as follows:

(1) preparation of explants: choose the bulbs of clematis as explants, rinse the explants with water for 20 minutes, put them into sodium hypochlorite for sterilization for 20 minutes, rinse with sterile water twice, drain the water, and use The scalpel cuts the surface sterilized explant to obtain the explant to be inoculated;

(2) Preparation of medium: According to the composition and dosage of MS basic medium, configure corresponding mother liquor, successively take the mother liquor of macroelements, the mother liquor of trace elements, the mother liquor of iron salt and the organic components, and after mixing, press the induction medium, proliferation and subculture The hormones are added to the base and components of the rooting medium.

Use a pH meter to measure the pH of the medium, and adjust the pH to 5.8 with 1M NaOH solution or 1M hydrochloric acid; then heat to dissolve the agar, and finally add water to make up the volume; divide the prepared medium into culture bottles and seal them with bottle caps. Sterilize by autoclaving at 121°C, and store in a clean area after solidification.

(3) induction culture: the sterilized clematis explants are inoculated on the induction medium, placed in the tissue culture room for induction culture, after culturing 20d, callus is obtained, and the edge of the bulb grows cluster buds;

Wherein, the induction medium is: sucrose 30g·L ^-1 + agar 5g·L ^-1 +MS+6-BA 2.0mg·L ^-1 +NAA1.0mg·L ^-1 +GA 1.5mg·L ^-1 , pH To 5.8, autoclave at 121°C; the conditions of induction culture are: temperature of 25°C, light of 12h/d, and light intensity of 1500lx.

(4) proliferation subculture: cut the induced clump buds, inoculate on the proliferation subculture medium, carry out the proliferation subculture, and transfer once every 25 days to obtain the clump seedlings;

Among them, the proliferation subculture medium is: sucrose 30g·L ^-1 + agar 5g·L ^-1 +MS+6-BA 1.5mg·L ^-1 +NAA0.5mg·L ^-1 , pH to 5.8, high pressure at 121°C Sterilization; the conditions of proliferation and subculture are: the temperature is 25°C, the light is 12h/d, and the light intensity is 1500lx.

(5) rooting culture: when the clump seedlings grow to 2～4cm, the clump seedlings are cut into individual plants, inoculated on the rooting medium, and the rooting culture is carried out, and after culturing for 25 days, a complete tissue culture seedling is obtained;

Among them, the rooting medium is: sucrose 30g·L ^-1 + agar 5g·L ^-1 +1/2MS+NAA 0.3mg·L ^-1 , pH to 5.8, autoclaved at 121°C; the conditions for rooting culture are: temperature The temperature is 25℃, the light is 12h/d, and the light intensity is 1500lx.

(6) Refining and transplanting: Take out the complete tissue culture seedlings after rooting culture, wash the culture medium of the roots, transplant them into the plug trays, and cultivate to obtain clematis.

Among them, the substrates are commonly used humus, perlite and pine bark. The upper layer of the greenhouse was covered with a shading net, and the ambient temperature for transplanting was 25 °C. Finally, the transplant survival rate was only 72%.

Comparative Example 6

During the tissue culture process of Clematis in this comparative example, the bacteriostatic agents of Comparative Examples 1 to 4 were respectively used, and the rest of the contents were exactly the same as those of Example 4.

Finally, the transplant survival rates were 75% (Comparative Example 1), 73% (Comparative Example 2), 78% (Comparative Example 4), and 80% (Comparative Example 4). The main bacterial species infected in clematis explants were Bacillus and Streptomyces (Comparative Examples 1-3), Pseudomonas and Rhodotorhinae (Comparative Example 4).

Claims (9)

1. a bacteriostatic agent for plant tissue culture, is characterized in that, in 1L, comprises the component of following consumption: Plant extract 100～500ml; Chitosan oligosaccharide 5～20g; Surfactant 5～20ml; the rest is water; The plant extract is an ethanol extract of a mixture of garlic bulb, ginger rhizome, bitter gourd peel and pomegranate peel. 2. plant tissue culture bacteriostatic agent as claimed in claim 1, is characterized in that, in 1L, comprises the component of following consumption: Plant extract 200ml; Chitooligosaccharide 20g; Surfactant 10ml; the rest is water; The plant extract is an ethanol extract of a mixture of garlic bulb, ginger rhizome, bitter gourd peel and pomegranate peel. 3. plant tissue culture bacteriostatic agent as claimed in claim 1 or 2, is characterized in that, the preparation method of described plant extract comprises: (1) after garlic bulb, ginger rhizome, balsam pear peel and pomegranate peel are mixed, add in ethanol, stir and pulverize to obtain mixed solution; (2) After the mixed solution is subjected to ultrasonic treatment, solid impurities are removed by filtration, and then ethanol is removed by evaporation and concentration to obtain a plant extract. 4. bacteriostatic agent for plant tissue culture as claimed in claim 3, is characterized in that, in step (1), the mass ratio of described garlic bulb, ginger rhizome, balsam pear peel and pomegranate peel is 1～3:1～ 2:1～2:1～2. 5 . The bacteriostatic agent for plant tissue culture according to claim 3 , wherein in step (2), the temperature of the ultrasonic treatment is 40-70° C., and the frequency is 30-50 KHz. 6 . 6. a kind of method utilizing the clematis tissue culture method that the plant tissue culture antibacterial agent as described in any one of claim 1～5 is carried out, it is characterized in that, comprising: (1) preparation of explant: get the explant of Clematis clematis, place it in the bacteriostatic agent for plant tissue culture after cleaning and soak, take out and rinse, obtain sterilized explant; (2) Induction culture: the sterilized explants are inoculated into the induction medium containing the bacteriostatic agent for plant tissue culture, and the induction culture is carried out to obtain callus containing clustered buds; (3) proliferation subculture: inoculate the callus containing the clump bud in the proliferation subculture medium containing the bacteriostatic agent for plant tissue culture, carry out the proliferation subculture, and obtain the clump seedling; (4) rooting culture: inoculate the clump seedlings in the rooting medium containing the bacteriostatic agent for the plant tissue culture, carry out rooting culture, and obtain tissue culture seedlings; (5) Refining and transplanting. 7. The method of claim 6, wherein the induction medium is MS+sucrose 20-30g·L ^-1 +agar 5-10g·L ^-1 +6 ^- BA 1.0-2.0mg·L- ¹ +NAA 0.2-1.0mg·L ^-1 +GA 1.5mg·L ^-1 +bacteriostat 200-300ml·L ^-1 . 8. The method of claim 6, wherein the proliferation subculture medium is MS+sucrose 20-30g·L ^-1 +agar 5-10g·L ^-1 +6-BA 0.5-1.5mg· L ^-1 +NAA 0.2-0.5mg·L ^-1 + bacteriostatic agent 300-400ml·L ^-1 . 9. method as claimed in claim 6 is characterized in that, described rooting medium is 1/2MS+sucrose 20-30g·L ^-1 +agar 5-10g·L ^{- 1} +NAA 0.3-0.5mg·L- ¹ + bacteriostatic agent 100-150ml·L ^-1 .

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