阅读说明：本技术 一种免灭菌条件的苹果组培抑菌剂及其制备方法 (Apple tissue culture bacteriostatic agent free of sterilization condition and preparation method thereof ) 是由 李强伟 张晓斌 谢金磊 王潇 孟祥勇 于 2021-07-12 设计创作，主要内容包括：本发明公开了一种免灭菌条件的苹果组培抑菌剂及其制备方法,涉及组培技术领域,通过在还原铁粉的催化还原下,使得对氯硝基苯中的硝基形成氨基,生成中间体1,之后中间体1与三乙胺发生季铵化反应,生成带有季铵基团的中间体2,之后中间体2与四氯乙烯亲核取代反应,合成中间体3,中间体3具有碳碳双键以及大量季铵基团,经过聚合后,形成高分子的抑菌剂；通过将该抑菌剂添加到苹果组培用的培养基中,达到无菌条件,无需高压灭菌,可以直接利用塑料杯代替组培瓶,简化组培环节,降低组培成本,同时,无菌条件温和,避免了培养基中的激素和营养元素的流失,保证了组培苗的营养吸收,使得组培苗生长健壮,分化率高。(The invention discloses an apple tissue culture bacteriostatic agent without sterilization conditions and a preparation method thereof, and relates to the technical field of tissue culture, wherein under the catalytic reduction of reduced iron powder, nitro in p-chloronitrobenzene forms amino to generate an intermediate 1, then the intermediate 1 and triethylamine are subjected to quaternization reaction to generate an intermediate 2 with a quaternary ammonium group, then the intermediate 2 and tetrachloroethylene are subjected to nucleophilic substitution reaction to synthesize an intermediate 3, the intermediate 3 has a carbon-carbon double bond and a large number of quaternary ammonium groups, and after polymerization, a high-molecular bacteriostatic agent is formed; the bacteriostatic agent is added into a culture medium for apple tissue culture, so that aseptic conditions are achieved, high-pressure sterilization is not needed, a plastic cup can be directly used for replacing a tissue culture bottle, tissue culture links are simplified, tissue culture cost is reduced, meanwhile, the aseptic conditions are mild, loss of hormones and nutrient elements in the culture medium is avoided, nutrient absorption of tissue culture seedlings is guaranteed, and the tissue culture seedlings grow robustly and have high differentiation rate.)

1. A preparation method of an apple tissue culture bacteriostatic agent without sterilization conditions is characterized by comprising the following steps:

the method comprises the following steps: adding reduced iron powder, glacial acetic acid and deionized water into a three-neck flask provided with a condensation reflux pipe, heating to boil and maintaining for 3-5min, then adding p-chloronitrobenzene into the three-neck flask, heating and refluxing for 0.5-2h, reacting completely, cooling in an ice water bath, adding sodium chloride, precipitating crystals, carrying out vacuum filtration, collecting a filter cake, placing the filter cake in a vacuum drying oven, and drying at the temperature of 60-80 ℃ to constant weight to obtain an intermediate 1;

step two: adding absolute ethyl alcohol, triethylamine and the intermediate 1 into a three-neck flask provided with a condensation reflux pipe and a stirrer, stirring and reacting for 8-10h at the temperature of 70-80 ℃ and the stirring speed of 100-300r/min, then distilling the reaction product under reduced pressure at the temperature of 60 ℃ to remove the solvent, washing the distillation product with ethyl acetate and acetone for 2-3 times respectively, carrying out vacuum filtration, placing the filter cake into a vacuum drying box, and drying at the temperature of 40-50 ℃ to constant weight to obtain an intermediate 2;

step three: adding tetrachloroethylene, the intermediate 2 and anhydrous ether into a three-neck flask provided with a constant-pressure dropping funnel, a condensation reflux pipe and a stirrer, dropwise adding a sodium hydroxide solution while stirring under the conditions of ice water bath and stirring speed of 200-500r/min, heating after dropwise adding, carrying out reflux reaction for 6-8h, adding deionized water into a reaction product after the reaction is finished, continuously stirring for 3-5min, extracting for 2-3 times by using ethyl acetate, combining extract liquor, drying by using anhydrous sodium sulfate, and carrying out rotary evaporation to remove solvent to obtain an intermediate 3;

step four: adding the intermediate 3 and deionized water into a four-neck flask provided with a thermometer, a constant-pressure dropping funnel, a stirrer and a condenser, stirring and dropwise adding a sodium hydroxide solution under the condition of stirring speed of 200-300r/min to adjust the pH value of a reaction system to 7-8, heating and carrying out reflux reaction, dropwise adding an ammonium persulfate solution under stirring, controlling the dropwise adding time to be 2-3h, continuously stirring and reacting for 1-2h after the dropwise adding is finished, adding a reaction product into a methanol aqueous solution to wash for 2-3 times, filtering, and drying a filter cake in vacuum to constant weight to obtain the apple tissue culture bacteriostatic agent under the sterilization-free condition.

2. The preparation method of the apple tissue culture bacteriostatic agent without the sterilization condition as claimed in claim 1, wherein the dosage ratio of the reduced iron powder, the glacial acetic acid, the deionized water and the p-chloronitrobenzene in the step one is 1-2 mol: 20mL of: 80mL of: 0.6 mol.

3. The preparation method of the apple tissue culture bacteriostatic agent without the sterilization condition as claimed in claim 1, wherein the dosage ratio of the absolute ethyl alcohol, the triethylamine and the intermediate 1 is 100 mL: 1.0 mol: 1.0-1.5 mol.

4. The preparation method of the apple tissue culture bacteriostatic agent without sterilization conditions as claimed in claim 1, wherein the dosage ratio of tetrachloroethylene, intermediate 2, anhydrous ether, sodium hydroxide solution and deionized water is 0.1 mol: 0.4: 100mL of: 20mL of: 100mL, and the molar concentration of the sodium hydroxide solution is 8-10 mol/L.

5. The preparation method of the apple tissue culture bacteriostatic agent under the sterilization-free condition according to claim 1, wherein the dosage ratio of the intermediate 3, deionized water and ammonium persulfate solution is 1 g: 10-20 mL: 15mL, wherein the molar concentration of the sodium hydroxide solution is 8-10mol/L, and the volume fraction of the methanol aqueous solution is 50-60%.

Technical Field

The invention relates to the technical field of tissue culture, in particular to an apple tissue culture bacteriostatic agent without sterilization conditions and a preparation method thereof.

Background

The tissue culture of plants is a new technology of asexual propagation developed in recent decades according to the theory that plant cells have totipotency, the tissue culture of plants is also called isolated culture in a broad sense, and means that tissues, organs or cells, protoplasts and the like which meet the requirements are separated from plant bodies, and the tissues, organs or cells, the protoplasts and the like are inoculated on a culture medium containing various nutrient substances and phytohormones to be cultured under the aseptic condition through aseptic operation so as to obtain regenerated complete plants or produce other products with economic value;

the tissue culture of the apple adopts an aseptic culture technology, and organs such as stem tips, axillary buds, leaves and the like from fine plants and tissue slices thereof are subjected to isolated culture, so that a large number of individuals with consistent heredity can be obtained in a short period;

therefore, the key point of the invention is to solve the above problems by requiring an apple tissue culture bacteriostatic agent without sterilization.

Disclosure of Invention

In order to overcome the technical problems, the invention aims to provide an apple tissue culture bacteriostatic agent without sterilization conditions and a preparation method thereof: the method is characterized in that under the catalytic reduction of reduced iron powder, nitro in p-chloronitrobenzene forms amino to generate an intermediate 1, then the intermediate 1 and triethylamine are subjected to quaternization reaction to generate an intermediate 2 with quaternary ammonium groups, then the intermediate 2 and tetrachloroethylene are subjected to nucleophilic substitution reaction to synthesize an intermediate 3, the intermediate 3 has carbon-carbon double bonds and a large number of quaternary ammonium groups, and the intermediate 3 is polymerized to increase the relative molecular weight and improve the charge density, so that the antibacterial property is greatly improved, thereby forming the high-molecular bacteriostatic agent.

The purpose of the invention can be realized by the following technical scheme:

an apple tissue culture bacteriostatic agent free of sterilization conditions has the following structure:

the preparation method of the apple tissue culture bacteriostatic agent without the sterilization condition comprises the following steps:

the method comprises the following steps: adding reduced iron powder, glacial acetic acid and deionized water into a three-neck flask provided with a condensation reflux pipe, heating to boil and maintaining for 3-5min, then adding p-chloronitrobenzene into the three-neck flask, heating and refluxing for 0.5-2h, reacting completely, cooling in an ice water bath, adding sodium chloride, precipitating crystals, carrying out vacuum filtration, collecting a filter cake, placing the filter cake in a vacuum drying oven, and drying at the temperature of 60-80 ℃ to constant weight to obtain an intermediate 1;

the reaction principle is as follows:

step two: adding absolute ethyl alcohol, triethylamine and the intermediate 1 into a three-neck flask provided with a condensation reflux pipe and a stirrer, stirring and reacting for 8-10h at the temperature of 70-80 ℃ and the stirring speed of 100-300r/min, then distilling the reaction product under reduced pressure at the temperature of 60 ℃ to remove the solvent, washing the distillation product with ethyl acetate and acetone for 2-3 times respectively, carrying out vacuum filtration, placing the filter cake into a vacuum drying box, and drying at the temperature of 40-50 ℃ to constant weight to obtain an intermediate 2;

the reaction principle is as follows:

step three: adding tetrachloroethylene, the intermediate 2 and anhydrous ether into a three-neck flask provided with a constant-pressure dropping funnel, a condensation reflux pipe and a stirrer, dropwise adding a sodium hydroxide solution while stirring under the conditions of ice water bath and stirring speed of 200-500r/min, heating after dropwise adding, carrying out reflux reaction for 6-8h, adding deionized water into a reaction product after the reaction is finished, continuously stirring for 3-5min, extracting for 2-3 times by using ethyl acetate, combining extract liquor, drying by using anhydrous sodium sulfate, and carrying out rotary evaporation to remove solvent to obtain an intermediate 3;

the reaction principle is as follows:

step four: adding the intermediate 3 and deionized water into a four-neck flask provided with a thermometer, a constant-pressure dropping funnel, a stirrer and a condenser, stirring and dropwise adding a sodium hydroxide solution under the condition of stirring speed of 200-300r/min to adjust the pH value of a reaction system to 7-8, heating and carrying out reflux reaction, dropwise adding an ammonium persulfate solution under stirring, controlling the dropwise adding time to be 2-3h, continuously stirring and reacting for 1-2h after the dropwise adding is finished, adding a reaction product into a methanol aqueous solution to wash for 2-3 times, filtering, and drying a filter cake in vacuum to constant weight to obtain the apple tissue culture bacteriostatic agent under the sterilization-free condition.

The reaction principle is as follows:

as a further scheme of the invention: the dosage ratio of the reduced iron powder, the glacial acetic acid, the deionized water and the p-chloronitrobenzene in the step one is 1-2 mol: 20mL of: 80mL of: 0.6 mol.

As a further scheme of the invention: the dosage ratio of the absolute ethyl alcohol to the triethylamine to the intermediate 1 is 100 mL: 1.0 mol: 1.0-1.5 mol.

As a further scheme of the invention: the dosage ratio of the tetrachloroethylene, the intermediate 2, the anhydrous ether, the sodium hydroxide solution and the deionized water is 0.1 mol: 0.4: 100mL of: 20mL of: 100mL, and the molar concentration of the sodium hydroxide solution is 8-10 mol/L.

As a further scheme of the invention: the using ratio of the intermediate 3 to deionized water to the ammonium persulfate solution is 1 g: 10-20 mL: 15mL, wherein the molar concentration of the sodium hydroxide solution is 8-10mol/L, and the volume fraction of the methanol aqueous solution is 50-60%.

The invention has the beneficial effects that:

the invention is that under the catalytic reduction of reduced iron powder, nitro in p-chloronitrobenzene forms amino to generate intermediate 1, then intermediate 1 and triethylamine are made to have quaternization reaction to generate intermediate 2 with quaternary ammonium group, then intermediate 2 and tetrachloroethylene are made to have nucleophilic substitution reaction to synthesize intermediate 3, intermediate 3 has carbon-carbon double bond and a large amount of quaternary ammonium group, the quaternary ammonium group is positively charged and can be absorbed on the surface of bacteria, penetrate cell wall, combine with cell membrane to disturb cell membrane composition, cause intracellular substance leakage, finally bacterial death, the carbon-carbon double bond makes it have polymerization condition, after polymerization, relative molecular weight is increased, charge density is increased, so antibacterial property is greatly improved, thus forming macromolecule bacteriostatic agent, therefore, the bacteriostatic agent has excellent antibacterial property, and is added into culture medium for apple tissue culture, the bacteria-free condition is achieved through the action of the bacteriostatic agent, high-pressure sterilization is not needed, the plastic cup can be directly used for replacing a tissue culture bottle, the tissue culture link is simplified, the tissue culture cost is reduced, meanwhile, the bacteria-free condition is mild, the loss of hormones and nutrient elements in a culture medium is avoided, the nutrient absorption of tissue culture seedlings is guaranteed, the tissue culture seedlings grow healthily and the differentiation rate is high.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1:

the embodiment is a preparation method of an apple tissue culture bacteriostatic agent under a sterilization-free condition, which comprises the following steps:

the method comprises the following steps: adding reduced iron powder, glacial acetic acid and deionized water into a three-neck flask provided with a condensation reflux pipe, heating to boil and maintaining for 3min, then adding p-chloronitrobenzene into the three-neck flask, heating and refluxing for 0.5h, completely reacting, cooling in an ice water bath, adding sodium chloride, separating out crystals, carrying out vacuum filtration, collecting a filter cake, placing the filter cake in a vacuum drying box, and drying at the temperature of 60 ℃ to constant weight to obtain an intermediate 1; controlling the dosage ratio of the reduced iron powder, the glacial acetic acid, the deionized water and the p-chloronitrobenzene to be 1 mol: 20mL of: 80mL of: 0.6 mol;

step two: adding absolute ethyl alcohol, triethylamine and the intermediate 1 into a three-neck flask provided with a condensing reflux pipe and a stirrer, stirring and reacting for 8 hours at the temperature of 70 ℃ and the stirring speed of 100r/min, then distilling the reaction product under reduced pressure at the temperature of 60 ℃ to remove a solvent, washing the distillation product with ethyl acetate and acetone for 2 times respectively, carrying out vacuum filtration, placing the filter cake in a vacuum drying oven, and drying at the temperature of 40 ℃ to constant weight to obtain an intermediate 2; controlling the dosage ratio of absolute ethyl alcohol, triethylamine and the intermediate 1 to be 100 mL: 1.0 mol: 1.0 mol;

step three: adding tetrachloroethylene, the intermediate 2 and anhydrous ether into a three-neck flask provided with a constant-pressure dropping funnel, a condensation reflux pipe and a stirrer, dropwise adding a sodium hydroxide solution while stirring under the conditions of ice water bath and stirring speed of 200r/min, heating after dropwise adding, carrying out reflux reaction for 6 hours, adding deionized water into a reaction product after the reaction is finished, continuously stirring for 3 minutes, extracting for 2 times by using ethyl acetate, combining extract liquor, drying by using anhydrous sodium sulfate, and carrying out rotary evaporation to remove solvent to obtain an intermediate 3; controlling the dosage ratio of tetrachloroethylene, the intermediate 2, anhydrous ether, sodium hydroxide solution and deionized water to be 0.1 mol: 0.4: 100mL of: 20mL of: 100mL, and the molar concentration of the sodium hydroxide solution is 8 mol/L;

step four: adding the intermediate 3 and deionized water into a four-neck flask provided with a thermometer, a constant-pressure dropping funnel, a stirrer and a condenser, stirring while dropwise adding a sodium hydroxide solution to adjust the pH of a reaction system to 7 under the condition of stirring speed of 200r/min, heating and carrying out reflux reaction, dropwise adding an ammonium persulfate solution while stirring, controlling the dropwise adding time to be 2 hours, continuously stirring to react for 1 hour after the dropwise adding is finished, adding a reaction product into a methanol aqueous solution to wash for 2 times, filtering, and carrying out vacuum drying on a filter cake to constant weight to obtain the apple tissue culture bacteriostatic agent under the sterilization-free condition; the using ratio of the intermediate 3 to deionized water to the ammonium persulfate solution is 1 g: 10mL of: 15mL, the molar concentration of the sodium hydroxide solution is 8mol/L, and the volume fraction of the methanol aqueous solution is 50%.

Example 2:

the embodiment is a preparation method of an apple tissue culture bacteriostatic agent under a sterilization-free condition, which comprises the following steps:

the method comprises the following steps: adding reduced iron powder, glacial acetic acid and deionized water into a three-neck flask provided with a condensation reflux pipe, heating to boil and maintaining for 4min, then adding p-chloronitrobenzene into the three-neck flask, heating and refluxing for 1h, completely reacting, cooling in an ice water bath, adding sodium chloride, separating out crystals, carrying out vacuum filtration, collecting a filter cake, placing the filter cake in a vacuum drying oven, and drying at the temperature of 70 ℃ to constant weight to obtain an intermediate 1; controlling the dosage ratio of the reduced iron powder, the glacial acetic acid, the deionized water and the p-chloronitrobenzene to be 1.5 mol: 20mL of: 80mL of: 0.6 mol;

step two: adding absolute ethyl alcohol, triethylamine and the intermediate 1 into a three-neck flask provided with a condensing reflux pipe and a stirrer, stirring and reacting for 9 hours at the temperature of 75 ℃ and the stirring speed of 200r/min, then distilling the reaction product under reduced pressure at the temperature of 60 ℃ to remove a solvent, washing the distillation product for 3 times respectively by using ethyl acetate and acetone, carrying out vacuum filtration, placing the filter cake in a vacuum drying oven, and drying the filter cake to constant weight at the temperature of 45 ℃ to obtain an intermediate 2; controlling the dosage ratio of absolute ethyl alcohol, triethylamine and the intermediate 1 to be 100 mL: 1.0 mol: 1.2 mol;

step three: adding tetrachloroethylene, the intermediate 2 and anhydrous ether into a three-neck flask provided with a constant-pressure dropping funnel, a condensation reflux pipe and a stirrer, dropwise adding a sodium hydroxide solution while stirring under the conditions of ice water bath and a stirring speed of 350r/min, heating after dropwise adding, carrying out reflux reaction for 7 hours, adding deionized water into a reaction product after the reaction is finished, continuously stirring for 4 minutes, extracting for 3 times by using ethyl acetate, combining extract liquor, drying by using anhydrous sodium sulfate, and carrying out rotary evaporation to remove solvent to obtain an intermediate 3; controlling the dosage ratio of tetrachloroethylene, the intermediate 2, anhydrous ether, sodium hydroxide solution and deionized water to be 0.1 mol: 0.4: 100mL of: 20mL of: 100mL, and the molar concentration of the sodium hydroxide solution is 9 mol/L;

step four: adding the intermediate 3 and deionized water into a four-neck flask provided with a thermometer, a constant-pressure dropping funnel, a stirrer and a condenser, stirring while dropwise adding a sodium hydroxide solution under the condition of a stirring speed of 250r/min to adjust the pH of a reaction system to 7.5, heating and carrying out reflux reaction, dropwise adding an ammonium persulfate solution while stirring, controlling the dropwise adding time to be 2.5h, continuously stirring and reacting for 1.5h after the dropwise adding is finished, adding a reaction product into a methanol aqueous solution to wash for 3 times, filtering, and carrying out vacuum drying on a filter cake to constant weight to obtain the apple tissue culture bacteriostatic agent under the sterilization-free condition; the using ratio of the intermediate 3 to deionized water to the ammonium persulfate solution is 1 g: 15mL of: 15mL, the molar concentration of the sodium hydroxide solution is 9mol/L, and the volume fraction of the methanol aqueous solution is 55%.

Example 3:

the embodiment is a preparation method of an apple tissue culture bacteriostatic agent under a sterilization-free condition, which comprises the following steps:

the method comprises the following steps: adding reduced iron powder, glacial acetic acid and deionized water into a three-neck flask provided with a condensation reflux pipe, heating to boil and maintaining for 5min, then adding p-chloronitrobenzene into the three-neck flask, heating and refluxing for 2h, completely reacting, cooling in an ice water bath, adding sodium chloride, separating out crystals, carrying out vacuum filtration, collecting a filter cake, placing the filter cake in a vacuum drying oven, and drying at the temperature of 80 ℃ to constant weight to obtain an intermediate 1; controlling the dosage ratio of the reduced iron powder, the glacial acetic acid, the deionized water and the p-chloronitrobenzene to be 2 mol: 20mL of: 80mL of: 0.6 mol;

step two: adding absolute ethyl alcohol, triethylamine and the intermediate 1 into a three-neck flask provided with a condensing reflux pipe and a stirrer, stirring and reacting for 10 hours at the temperature of 80 ℃ and the stirring speed of 300r/min, then distilling the reaction product under reduced pressure at the temperature of 60 ℃ to remove a solvent, washing the distillation product with ethyl acetate and acetone for 3 times respectively, carrying out vacuum filtration, placing the filter cake in a vacuum drying oven, and drying at the temperature of 50 ℃ to constant weight to obtain an intermediate 2; controlling the dosage ratio of absolute ethyl alcohol, triethylamine and the intermediate 1 to be 100 mL: 1.0 mol: 1.5 mol;

step three: adding tetrachloroethylene, the intermediate 2 and anhydrous ether into a three-neck flask provided with a constant-pressure dropping funnel, a condensation reflux pipe and a stirrer, dropwise adding a sodium hydroxide solution while stirring under the conditions of ice water bath and stirring speed of 500r/min, heating after dropwise adding, carrying out reflux reaction for 8 hours, adding deionized water into a reaction product after the reaction is finished, continuously stirring for 5 minutes, extracting for 3 times by using ethyl acetate, combining extract liquor, drying by using anhydrous sodium sulfate, and carrying out rotary evaporation to remove solvent to obtain an intermediate 3; controlling the dosage ratio of tetrachloroethylene, the intermediate 2, anhydrous ether, sodium hydroxide solution and deionized water to be 0.1 mol: 0.4: 100mL of: 20mL of: 100mL, and the molar concentration of the sodium hydroxide solution is 10 mol/L;

step four: adding the intermediate 3 and deionized water into a four-neck flask provided with a thermometer, a constant-pressure dropping funnel, a stirrer and a condenser, stirring while dropwise adding a sodium hydroxide solution under the condition of stirring speed of 300r/min to adjust the pH of a reaction system to 8, heating and carrying out reflux reaction, dropwise adding an ammonium persulfate solution while stirring, controlling the dropwise adding time to be 3h, continuing stirring and reacting for 2h after the dropwise adding is finished, adding a reaction product into a methanol aqueous solution, washing for 3 times, filtering, and carrying out vacuum drying on a filter cake to constant weight to obtain the apple tissue culture bacteriostatic agent under the sterilization-free condition; the using ratio of the intermediate 3 to deionized water to the ammonium persulfate solution is 1 g: 20mL of: 15mL, the molar concentration of the sodium hydroxide solution is 10mol/L, and the volume fraction of the methanol aqueous solution is 60%.

Comparative example 1:

comparative example 1 is plant tissue culture antimicrobial PPM.

Comparative example 2:

comparative example 2 is a bacteriostatic agent for the tissue culture medium of seedling under application No. CN 201410781040.4.

The bacteriostatic agents of examples 1-3 and comparative examples 1-2 were prepared into 10% bacteriostatic solutions with a mass fraction of 1%, and each 3mL of the bacteriostatic solution was added to a plate and mixed with 10mL of nutrient agar medium (10 g/L peptone, 3g/L beef extract powder, 5g/L sodium chloride, 15g/L agar, final pH7.3-7.5) to prepare a bacteriostatic plate. The test germs (mixed germs of Escherichia coli and Staphylococcus aureus) were cultured in advance in nutrient agar medium (peptone 10g/L, beef extract powder 3g/L, sodium chloride 5g/L, agar 15g/L, final pH7.3-7.5), and the uniformly grown germs were cut out at the colony edge with a punch having a pore diameter of 0.5cm as an inoculum. The inoculum was transferred to the center of the bacteriostatic plate. Sterile distilled water was used as a control, and the culture was carried out at a constant temperature of 25 ℃. The colony diameter was measured by the cross method and corrected to calculate the percent inhibition. The bacteriostatic rate (%) × (control colony diameter-treated colony diameter)/(control colony diameter) × 100%. The results are shown in the following table:

referring to the data in the table, according to the comparison between the examples and the comparative examples 1-2, it can be seen that the bacteriostatic agent of the present invention is more suitable for the apple tissue culture technology than the bacteriostatic agent in the prior art, and has a better bacteriostatic effect.

In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

The foregoing is illustrative and explanatory only and is not intended to be exhaustive or to limit the invention to the precise embodiments described, and various modifications, additions, and substitutions may be made by those skilled in the art without departing from the scope of the invention or exceeding the scope of the claims.