CN116941533A

CN116941533A - Pepper anther culture method

Info

Publication number: CN116941533A
Application number: CN202311155349.8A
Authority: CN
Inventors: 张丁; 张大庆; 李展; 张丹丹; 华松
Original assignee: Dangshan Shenqing Seed Industry Co ltd
Current assignee: Dangshan Shenqing Seed Industry Co ltd
Priority date: 2023-09-08
Filing date: 2023-09-08
Publication date: 2023-10-27

Abstract

The invention discloses a pepper anther culture method, and relates to the technical field of pepper anther culture; the method comprises the following steps: planting the capsicum to be tested in a farmland, and spraying systemic pesticide after bud emergence; the flower buds of the plants without virus and insect damage are selected to be placed in self-sealing bags, and then are transferred into an ice box; placing the ice box with the flower buds in a refrigerator at 1-5 ℃ for pretreatment for 1-3 days; then placing the flower buds into a 0.25% sodium hypochlorite solution for surface sterilization for 10-15min, then flushing with sterile water for 3-4 times, and pouring the flower buds onto sterile filter paper; the petals are picked up by forceps, pressed, anthers on the petals naturally fall off on the surface of the induction culture medium, and then the induction culture medium is sealed by a membrane. According to the invention, hormone treatment is added in the cultivation process, so that the induction rate of the tissues in distress and the induction rate of embryoids of the peppers are greatly increased, the anti-pollution capability of the peppers is increased, and the survival rate is increased.

Description

Pepper anther culture method

Technical Field

The invention relates to the technical field of pepper anther culture, in particular to a pepper anther culture method.

Background

Capsicum (capsicum nuum l.) is an important commercial crop in the world and can be used as vegetables, spices, food pigments and medicines, and as artificial breeding technology is mature, pepper anther culture can shorten the cultivation period, increase the breeding yield and the like, and has been widely used.

For example, by searching, chinese patent publication No.: CN103444542B discloses a method for tissue culture of pepper anther, comprising: (1) selecting flower buds with microspores in a mononuclear borderline period; (2) Soaking the flower buds in 70% alcohol for 0.5-1 min, washing with 12%o mercuric chloride for 8-15 min, washing with sterile water for 3-5 times, and sucking the water with sterile filter paper to obtain sterile anther in a single-core borderline period; (3) Floating the sterile anther in the single-nucleus borderline period in a liquid culture medium, and performing shake culture in darkness for 20-25 days, and then performing shake culture in illumination; (4) And (3) after 10-20 days of light shake culture, transferring the formed cotyledon embryo into an MSO solid culture medium for culture, and growing into a normal plant.

The above patent suffers from the following disadvantages: the strain is easy to be polluted by fungi and bacteria during the early spore culture and anther culture, so that the pollution rate and death rate of the capsicum are increased, and the survival rate of final culture is reduced.

Therefore, the invention provides a pepper anther culture method.

Disclosure of Invention

The invention aims to solve the defects in the prior art and provides a pepper anther culture method.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

a pepper anther culture method comprises the following steps:

s1: planting the capsicum to be tested in a farmland, and spraying systemic pesticide after bud emergence;

s2: the flower buds of the plants without virus and insect damage are selected to be placed in self-sealing bags, and then are transferred into an ice box;

s3: placing the ice box with the flower buds in a refrigerator at 1-5 ℃ for pretreatment for 1-3 days;

s4: then placing the flower buds into a 0.25% sodium hypochlorite solution for surface sterilization for 10-15min, then flushing with sterile water for 3-4 times, and pouring the flower buds onto sterile filter paper;

s5: the petals are picked up by forceps, pressed, anthers on the petals naturally fall off on the surface of the induction culture medium, and then the induction culture medium is sealed by a membrane;

s6: placing the sealed culture medium into a biochemical incubator at 25-30 ℃ for induction culture for 4-5 weeks under dark condition;

s7: stripping embryoid, inoculating into a sugar-free tissue culture box, introducing carbon dioxide, and performing micro-light autotrophic micropropagation;

s8: after domestication, transplanting the capsicum into a seedling training matrix, and after the capsicum grows stably, dipping 0.25% colchicine into absorbent cotton for chromosome doubling treatment for 36-50 hours.

Preferably: in the step S2, the bud picking time is 6 from April No. 1 to April No. 23: 00-9:30, selecting flower buds with petals equal to the calyx or purple according to 1/3 of the anther color.

Preferably: the basic culture medium comprises major element components 4120-4940, trace element components 35.35-41.12, ferric salt 61.05-69.1, organic components 81.65-124.55, sucrose 28000-32000 and agar 6800-7200;

the microelements comprise the following components in percentage by mass: potassium iodide 0.8-0.86, boric acid 6.0-6.4, manganese sulfate 20.3-24.3, zinc sulfate 8-9.2, sodium molybdate 0.2-0.3, copper sulfate 0.020-0.030, and cobalt chloride 0.020-0.030;

the ferric salt consists of the following components in percentage by mass: 35.25-39.25 parts of disodium ethylenediamine tetraacetate and 25.8-29.85 parts of ferrous sulfate;

the organic component comprises the following components in percentage by mass: 80-120 parts of inositol, 1-3 parts of glycine, 0.05-0.15 part of thiamine hydrochloride, 60.3-0.7 part of pyridoxine hydrochloride VB, and 0.3-0.7 part of nicotinic acid VB5 or VPP;

the hormone component is any one of a hormone component I, a hormone component II and a hormone component III.

Preferably: the hormone component I consists of the following components in percentage by mass: KT culture medium 0.3-0.7 and NAA culture medium 2.0-3.0;

the hormone component II consists of the following components in percentage by mass: IAA culture medium 0.1-0.3, KT culture medium 0.3-0.7;

the hormone component III consists of the following components in percentage by mass: 2,4-D culture medium 0.05-0.15, KT culture medium 0.3-0.7.

Preferably: in the step S5, the induction culture medium further comprises a bacteriostat, wherein the bacteriostat comprises benzyl amine penicillin and amphotericin B, and the amphotericin B is water-insoluble amphotericin B.

The preparation method of the bacteriostat comprises the following steps:

a1: firstly, putting the benzyl amine penicillin and the amphotericin B into a culture medium, and slowly stirring to dissolve the benzyl amine penicillin and the amphotericin B;

a2: then dimethyl sulfoxide is added as a cosolvent, and the cosolvent is added into the culture medium and then stirred to be dissolved;

a3: then ammonia gas is introduced into the culture medium, and the culture medium is slowly stirred until amphotericin B is dissolved.

Preferably: in the step S5, the induction culture medium further comprises a bacteriostat, wherein the bacteriostat comprises cephalosporin and amphotericin B, and the amphotericin B is water-insoluble amphotericin B.

The preparation method of the bacteriostat comprises the following steps:

a1: firstly, putting cephalosporin and amphotericin B into a culture medium, and slowly stirring to dissolve the cephalosporin and amphotericin B;

Preferably: in the step S5, the induction culture medium further comprises a bacteriostat, wherein the bacteriostat comprises benzyl amine penicillin, amphotericin B and cephalosporin, and the amphotericin B is water-insoluble amphotericin B.

The preparation method of the bacteriostat comprises the following steps:

a1: firstly, putting the benzyl amine penicillin and the amphotericin B and the cephalosporin into a culture medium, and slowly stirring to dissolve the mixture;

Preferably: in S7, the specific conditions are: illuminating for 14 hours each day; the illumination intensity is 2500 Lux-3000 Lux; the temperature is 23-26 ℃; the humidity is 80% -92%.

Preferably: in the step S7, the sugar-free tissue culture box comprises a box body, a support frame fixed in the lower part of the box body and a box cover connected to the top of the box body, wherein an aeration pipe is fixed at the bottom of the box body and is formed by mutually welding and communicating a plurality of annular pipes and straight pipes, the bottoms of the annular pipes and the straight pipes are provided with aeration holes, and the aeration pipe is connected to a gas supply mechanism through an air inlet pipe;

the gas supply mechanism comprises a carbon dioxide storage tank, an oxygen storage tank and a variable frequency pump, wherein the carbon dioxide storage tank and the oxygen storage tank are connected with a transposition valve through a pipeline, the transposition valve is connected with the gas inlet end of the variable frequency pump through a three-way pipe, and the gas outlet end of the variable frequency pump is connected with the gas inlet pipe through a pipeline;

the transposition valve comprises a valve body, and a first valve core and a second valve core which are connected in the valve body in a sliding manner, wherein the first valve core is connected with the second valve core through a connecting piece, two parallel air passage channels are formed in the inner wall of the valve body, and one air passage channel is formed in each of the inner walls of the first valve core and the second valve core; the side wall of the valve core II is connected with the valve body through a spring II, the side wall of the valve core I and the inner wall of the valve body are respectively fixed with a transposition electromagnetic valve, and the magnetic poles of the opposite sides of the two transposition electromagnetic valves are opposite;

the connecting piece comprises a flexible pad and a plurality of circular array elastic rods embedded in the flexible pad, wherein two sides of the flexible pad and the elastic rods are respectively fixed on the outer walls of one opposite side of the first valve core and the second valve core, and the elastic rods are of arc-shaped structures bent outwards.

Preferably: an insulating counterweight film frame is longitudinally and slidably connected to the inside of the box cover, and a breathable cotton spinning film is fixed inside the insulating counterweight film frame;

the top of the insulating counterweight film frame is fixedly provided with a resistance column, the inner wall of the box cover is embedded into an insulating sleeve, the inner side wall of the insulating sleeve is fixedly provided with a spring electrode, the spring electrode is in contact fit with the resistance column, and the spring electrode and the resistance column are connected in series to be connected into a power supply circuit of the variable frequency pump;

the breathable cotton spinning film is characterized in that notches are formed in two ends of the same diameter of the breathable cotton spinning film, film electrodes are contacted and fixed in the notches, locking assemblies in circular arrays are arranged on the side walls of the insulating counterweight film frames, locking electromagnetic valves are fixed on the inner walls of the box covers, which are positioned on the insulating counterweight film frames, and the film electrodes are connected in series to a power supply circuit of the locking electromagnetic valves;

the locking assembly comprises a permanent magnet which is connected with the insulating weight film frame in a sliding way and an anti-slip pad which is adhered to the side wall of the permanent magnet, the outer wall of the other side of the permanent magnet is connected with the inner wall of the insulating weight film frame through a first spring, and the magnetic poles of the permanent magnet and the opposite side of the locking electromagnetic valve are opposite.

The beneficial effects of the invention are as follows:

1. according to the invention, hormone treatment is added in the cultivation process, so that the induction rate of the tissues in distress and the induction rate of embryoids of the peppers are greatly increased, the anti-pollution capability of the peppers is increased, and the survival rate is increased.

2. According to the invention, the bacteriostatic agent is added to the culture medium, so that the survival of bacteria and fungi can be inhibited, and the pepper pollution is prevented, and the bacteriostatic ability of the culture medium is improved by combining the bacteriostatic agent, so that the survival rate is further increased.

3. According to the invention, on the basis of using amphotericin B, the water-solubility characteristic of amphotericin B is utilized to assist dissolution of insoluble amphotericin B in preparation, so that the amphotericin B has stronger antibacterial property compared with the soluble amphotericin B.

4. According to the invention, the aeration pipe is arranged, the gas supply mechanism is used for supplying the gas environment required by photosynthesis and respiration after chlorophyll appears in the embryoid, the annular pipe is composed of a plurality of annular pipes and linear pipes, so that aeration is more uniform, in addition, after gas is sprayed out of the aeration pipe, the aeration pipe is positioned at the bottom, and in the process of rising from the bottommost part of the nutrient solution in the box body, the aeration pipe can play a certain stirring function on the nutrient solution, so that nutrient components of the nutrient solution are distributed more uniformly, and sedimentation can be prevented.

5. The connecting piece is composed of the flexible pad on the outer side and the elastic rod on the inner side, so that the connecting piece can serve as acting force transmission medium between the first valve core and the second valve core on one hand, and can realize the mutual sealing function of the two gas channels of the valve body by utilizing the deformation characteristic of the elastic rod after position switching by combining the driving relation and the driven relation of the first valve core and the second valve core when the positions of the first valve core and the second valve core are changed on the other hand.

6. According to the invention, the spring electrode and the resistor column are arranged, and the circuit design is skillfully utilized, so that the combination of the spring electrode and the resistor column is similar to the structure of a sliding rheostat, the actual power of the variable frequency pump and the gas discharge speed are in a negative feedback adjusting chain, the waste of gas is prevented, and the cost is reduced.

7. According to the invention, the locking assembly is arranged, and according to the characteristics of the breathing action and photosynthesis of the peppers, the characteristics of the contact position of the pepper culture and the nutrient solution and the characteristics of spraying from the inside of the nutrient solution when the gas supply is combined, the wet gas supplied by the gas can be classified from the dry gas of the photosynthesis and the breathing action, and the ionic conduction characteristic of the nutrient solution is combined, so that the negative feedback control process can accurately detect the gas supply speed, and can prevent the influence of the gas generated by the self action of the peppers, thereby increasing the control precision.

8. According to the invention, the photovoltaic plate is additionally arranged, the photovoltaic plate can provide electric energy for the transposition electromagnetic valve through the photoelectric effect, and the photovoltaic plate and the sugar-free tissue culture box are in the same illumination environment, so that the automatic switching of gas supply can be realized by utilizing the environment variables of photosynthesis and respiration-illumination in the later period of sugar-free tissue culture.

Drawings

FIG. 1 is a schematic flow chart of a method for culturing pepper anther;

FIG. 2 is a schematic overall sectional view of a sugar-free tissue culture cassette for a pepper anther culture method according to the present invention;

FIG. 3 is a schematic view of an aeration tube of a sugar-free tissue culture box of the pepper anther culture method;

fig. 4 is a schematic cross-sectional view of a cover of a sugar-free tissue culture box according to the method for culturing pepper anther;

FIG. 5 is a schematic diagram II of an aeration pipe of a sugar-free tissue culture box of the pepper anther culture method;

FIG. 6 is an enlarged schematic view of part A of a sugar-free tissue culture box of the pepper anther culture method provided by the invention;

FIG. 7 is an enlarged schematic view of part B of a sugar-free tissue culture box of the pepper anther culture method provided by the invention;

FIG. 8 is a schematic view of a partial cross section of a sugar-free tissue culture cassette for a pepper anther culture method according to the present invention;

FIG. 9 is a schematic diagram showing the connection of the gas supply mechanism of the sugar-free tissue culture box in the method for cultivating the anther of capsicum according to the present invention;

FIG. 10 is a schematic cross-sectional view of a transposition valve of a sugar-free tissue culture box of the pepper anther culture method;

FIG. 11 is a diagram showing the circuit connection of the transposition electromagnet of the sugar-free tissue culture box in the pepper anther culture method;

FIG. 12 is an enlarged schematic view of part C of a sugar-free tissue culture cassette of a pepper anther culture method according to the present invention;

fig. 13 is a schematic diagram of the circuit connection of the variable frequency pump of the sugar-free tissue culture box in the pepper anther culture method;

fig. 14 is a schematic circuit connection diagram of a locking electromagnet of a sugar-free tissue culture box in the pepper anther culture method.

In the figure: 1-box body, 2-box cover, 3-support frame, 4-aeration pipe, 5-annular pipe, 6-straight line pipe, 7-intake pipe, 8-aeration hole, 9-ventilative cotton spinning film, 10-insulating counter weight film frame, 11-locking subassembly, 12-membrane electrode, 13-locking electro-magnet, 14-slipmat, 15-permanent magnet, 16-spring one, 17-insulating cover, 18-spring electrode, 19-resistance column, 20-carbon dioxide bin, 21-oxygen bin, 22-transposition valve, 23-tee pipe, 24-variable frequency pump, 25-photovoltaic board, 26-valve body, 27-spring two, 28-transposition electro-magnet, 29-case one, 30-connecting piece, 31-case two, 32-flexible mat, 33-elastic rod.

Detailed Description

The technical scheme of the patent is further described in detail below with reference to the specific embodiments.

In the description of this patent, it should be noted that, unless explicitly stated and limited otherwise, the terms "mounted," "connected," and "disposed" are to be construed broadly, and may be fixedly connected, disposed, detachably connected, disposed, or integrally connected, disposed, for example. The specific meaning of the terms in this patent will be understood by those of ordinary skill in the art as the case may be.

Example 1:

a pepper anther culture method comprises the following steps:

s3: placing the ice chest with the flower buds at 1-5 ℃, preferably 3 ℃ in the embodiment, and pre-treating in a refrigerator for 1-7 days;

s4: then placing the flower bud into 0.25% sodium hypochlorite solution for surface sterilization for 10-15min (preferably 12 min), then washing with sterile water for 3-4 times, and pouring onto sterile filter paper;

s5: the slide plate is picked up by forceps, petals are pressed, anthers on the petals naturally fall off the surface of the induction culture medium, and then the induction culture medium is sealed by a membrane;

s6: placing the sealed culture medium into a biochemical incubator at 25-30 ℃ and preferably 28 ℃ in the embodiment, and performing induction culture for 4-5 weeks under dark conditions;

s7: stripping embryoid, inoculating into a sugar-free tissue culture box, introducing carbon dioxide, and performing micro-light autotrophic micropropagation under certain conditions;

s8: transplanting the domesticated pepper to a seedling hardening matrix, dipping 0.25% colchicine into absorbent cotton to perform chromosome doubling treatment for 36-50 h after the pepper grows stably, wherein the preferred time is 48h in the embodiment.

In the step S2, the bud picking time is 16 on sunny days: 00-18:00, flower bud selection petals are equal in length with the calyx or in the S5 according to the flower buds with the 1/3 purple anther color, and an induction culture medium consists of a basic culture medium and hormone components.

The basic culture medium comprises a major element component 4530, a trace element component 38.23, an iron salt 65.1, an organic component 103.1, sucrose 30000 and agar 7000.

The macroelement component consists of the following components in percentage by mass: potassium nitrate 1900, ammonium nitrate 1650, potassium dihydrogen phosphate 170, magnesium sulfate 370, and calcium chloride 440.

The microelements comprise the following components in percentage by mass: potassium iodide 0.83, boric acid 6.2, manganese sulfate 22.3, zinc sulfate 8.6, sodium molybdate 0.25, copper sulfate 0.025, and cobalt chloride 0.025.

The ferric salt consists of the following components in percentage by mass: disodium ethylenediamine tetraacetate 37.25 and ferrous sulfate 27.85.

The organic component comprises the following components in percentage by mass: inositol 100, glycine 2, thiamine hydrochloride 0.1, pyridoxine hydrochloride VB60.5, niacin VB5 or VPP0.5.

The hormone component adopts a hormone component I and comprises the following components in percentage by mass: KT medium 0.5, NAA medium 2.5.

Example 2:

a method for culturing pepper anther, the present example is basically the same as example 1, except that: the hormone component adopts a hormone component II, and consists of the following components in percentage by mass: IAA medium 0.2, KT medium 0.5.

Example 3:

a method for culturing pepper anther, the present example is basically the same as example 1, except that: the hormone component adopts a hormone component III and consists of the following components in percentage by mass: 2,4-D medium 0.1, KT medium 0.5.

The following table shows the effects of different hormone components on pepper growth in examples 1-3

	Callus induction rate (%)	Embryoid induction rate (%)
			Example 1	43.69	5.25
Example 2	25.36	3.85
			Example 3	32.85	4.45
Hormone-free	10.35	0.36

As is clear from the above table, the hormone treatment added in examples 1 to 3 of the present invention significantly improves the induction rate of the pepper callus and the induction rate of embryoid bodies relative to the hormone-free treatment, and is optimal for the induction rate of the callus and the induction rate of embryoid bodies in example 1.

Example 4:

a pepper anther culture method is improved on the basis of examples 1-3, wherein the induction medium further comprises a bacteriostat, and the bacteriostat comprises benzyl amine penicillin and cephalosporin.

Example 5:

the pepper anther culture method is improved on the basis of the embodiment 1-3, the induction culture medium further comprises a bacteriostat, the bacteriostat comprises benzyl amine penicillin and amphotericin B, and the amphotericin B is water-insoluble amphotericin B.

The preparation method of the bacteriostat comprises the following steps:

Example 6:

the pepper anther culture method is improved on the basis of the embodiment 1-3, the induction culture medium further comprises a bacteriostat, the bacteriostat comprises cephalosporin and amphotericin B, and the amphotericin B is water-insoluble amphotericin B.

The preparation method of the bacteriostat comprises the following steps:

Example 7:

a pepper anther culture method is improved on the basis of the embodiment 1-3, the induction culture medium further comprises a bacteriostat, the bacteriostat comprises benzyl amine penicillin, amphotericin B and cephalosporin, and the amphotericin B is water-insoluble amphotericin B.

The preparation method of the bacteriostat comprises the following steps:

Comparative example 1:

a pepper anther culture method is improved on the basis of the embodiment 1-3, the induction culture medium further comprises a bacteriostat, the bacteriostat comprises benzyl amine penicillin, amphotericin B and cephalosporin, and the amphotericin B is amphotericin B which is easy to dissolve in water.

Comparative example 2:

The preparation method of the bacteriostat comprises the following steps: the benzyl amine penicillin and amphotericin B and cephalosporin are placed in a culture medium and slowly stirred to dissolve.

Table 2 is a comparison table of the contamination rate and mortality rate of the peppers by the different bacteriostats:

as shown in the table above, the addition of the bacteriostatic agent can increase the bacteriostatic ability of the culture medium, so that the pollution rate and the anther death rate of the culture medium can be reduced, and when the bacteriostatic agent adopts the combination of benzyl amine penicillin and amphotericin B and cephalosporin, the antibacterial property is strongest, and the pollution rate and the death rate are greatly improved.

Table 3: comparative table of contamination rate and mortality rate of capsicum using different types of amphotericin B and different preparation methods:

as shown in the table, when the bacteriostatic agent still adopts benzyl amine penicillin+amphotericin B+cephalosporin, but amphotericin B is of a type which is easy to dissolve in water, the bacteriostatic ability of the amphotericin B is greatly reduced, even the bacteriostatic ability is not as good as that of other bacteriostatic agents, so that the pollution rate and the death rate are greatly increased, when amphotericin B is of amphotericin B which is difficult to dissolve in water, but ammonia water and dimethyl sulfoxide are added for assisting dissolution, the bacteriostatic ability of the whole culture medium is still weaker, but when ammonia water and dimethyl sulfoxide are added for assisting dissolution, the solubility of the amphotericin B can reach more than 95%, so that the bacteriostatic ability of the whole culture medium is greatly increased, and the pollution rate and the death rate are greatly reduced.

From the above, the invention adopts the benzyl amine penicillin + amphotericin B + cephalosporin, and selects amphotericin B, and adopts ammonia water and dimethyl sulfoxide to improve the indissolvable property of amphotericin B, thereby greatly improving the bacteriostasis capability of the whole culture medium and greatly reducing the pollution rate and death rate.

Example 8:

the following modifications were made on the basis of examples 1 to 4 in this example: in S7, the specific conditions are: illuminating for 14 hours each day; the illumination intensity is 2500 Lux-3000 Lux; the temperature is 23-26 ℃; the humidity is 80% -92%.

Example 9:

in this embodiment, as shown in fig. 2 to 14, the sugar-free tissue culture cassette used in step S7 is not limited, and preferably: in the step S7, the sugar-free tissue culture box comprises a box body 1, a support frame 3 fixed in the lower part of the box body 1 and a box cover 2 connected to the top of the box body 1, wherein an aeration pipe 4 is fixed at the bottom of the box body 1, the aeration pipe 4 is formed by mutually welding and communicating a plurality of annular pipes 5 and linear pipes 6, aeration holes 8 are formed in the bottoms of the annular pipes 5 and the linear pipes 6, and the aeration pipe 4 is connected to a gas supply mechanism through an air inlet pipe 7; carbon dioxide can be supplied into the air inlet pipe 7 through the air supply mechanism to provide photosynthesis or oxygen can be supplied into the air inlet pipe 7 to provide respiration, and air enters the annular pipe 5 and the linear pipe 6 through the air inlet pipe 7 so as to be sprayed out through the aeration holes 8, and then the air is upwards moved and sprayed out from the nutrient solution in the box body 1 to be supplied to the peppers.

The device utilizes gas supply mechanism to supply the gas environment required by photosynthesis and respiration after chlorophyll appears in embryoid through setting up aeration pipe 4 to annular pipe 5 comprises a plurality of annular pipes 5 and sharp pipe 6, can make the aeration more even, and in addition, aeration hole 8 is located the bottom, and after the gas of its internal blowout, by the in-process of rising of nutrient solution bottommost in box body 1, can make it play certain "stirring" function to the nutrient solution for nutrient solution nutrient composition distributes more evenly, also can prevent to deposit.

In order to solve the problem of gas supply, as shown in fig. 9, the gas supply mechanism comprises a carbon dioxide storage tank 20, an oxygen storage tank 21 and a variable frequency pump 24, wherein the carbon dioxide storage tank 20 and the oxygen storage tank 21 are connected with a transposition valve 22 through a pipeline, the transposition valve 22 is connected with the air inlet end of the variable frequency pump 24 through a three-way pipe 23, and the air outlet end of the variable frequency pump 24 is connected with the air inlet pipe 7 through a pipeline; the transposition valve 22 can control the passage of the carbon dioxide storage tank 20 and the oxygen storage tank 21, when the photosynthesis needs the carbon dioxide supply, the carbon dioxide storage tank 20 can be communicated with the three-way pipe 23, the variable frequency pump 24 is started to suck the carbon dioxide into the air inlet pipe 7, when the respiration needs the useful oxygen supply, the transposition valve 22 communicates the oxygen storage tank 21 with the three-way pipe 23, and the variable frequency pump 24 is started to suck the oxygen into the air inlet pipe 7.

In order to solve the problem of path change, as shown in fig. 10, the transposition valve 22 comprises a valve body 26, and a first valve core 29 and a second valve core 31 which are slidably connected inside the valve body 26, wherein the first valve core 29 and the second valve core 31 are connected through a connecting piece 30, two parallel air path channels are formed in the inner wall of the valve body 26, and one air path channel is formed in each of the inner walls of the first valve core 29 and the second valve core 31; the side wall of the valve core II 31 is connected to the valve body 26 through a spring II 27, the side wall of the valve core I29 and the inner wall of the valve body 26 are respectively fixed with a transposition electromagnetic valve 28, and the magnetic poles of the opposite sides of the two transposition electromagnetic valves 28 are opposite; when the transposition solenoid valve 28 is electrified, a magnetic field is generated, the magnetic fields of the two transposition solenoid valves 28 repel each other, so that the valve core I29 and the valve core II 31 repel each other to move leftwards, the spring II 27 contracts, the gas channel of the valve core II 31 is communicated with one of the gas channels of the valve body 26, and when the transposition solenoid valve 28 is powered off, the valve core II 31 moves rightwards under the action of the elasticity of the spring II 27, so that the gas channel of the valve core I29 is communicated with the other gas channel of the valve body 26.

In order to solve the problem of mutual sealing of two gas channels, as shown in fig. 12, the connecting piece 30 includes a flexible pad 32 and a plurality of circular array elastic rods 33 embedded in the flexible pad 32, where two sides of the flexible pad 32 and the elastic rods 33 are respectively fixed on outer walls of opposite sides of the first valve core 29 and the second valve core 31, the elastic rods 33 are in an arc structure bent outwards, when the first valve core 29 is driven by the transposition solenoid valve 28, after the second valve core 31 is pressed to reach the end, due to the action force between the first valve core 29 and the second valve core 31, the elastic rods 33 are elastically deformed and bent outwards, so that the flexible pad 32 is tightly attached to the inner wall of the valve body 26, and when the second valve core 31 is driven by the second spring 27, after the first valve core 29 reaches the end for several days, due to the action force between the first valve core 29 and the second valve core 31, the elastic rods 33 are elastically deformed and bent outwards, so that the flexible pad 32 is tightly attached to the inner wall of the valve body 26.

The device is provided with the connecting piece 30, which is composed of the flexible pad 32 on the outer side and the elastic rod 33 on the inner side, and can be used as acting force transmission medium between the first valve core 29 and the second valve core 31, and on the other hand, the driving and driven relations of the first valve core 29 and the second valve core 31 when the positions are changed are combined, so that after the positions are switched, the mutual sealing function of two gas channels of the valve body 26 can be realized by utilizing the deformation characteristic of the elastic rod 33.

In order to solve the problem of controlling the gas supply speed, as shown in fig. 4-14, an insulating weight film frame 10 is longitudinally and slidably connected to the inside of the box cover 2, and a breathable cotton spinning film 9 is fixed to the inside of the insulating weight film frame 10; when the air is introduced, the air in the box body 1 is discharged through the air-permeable cotton spinning film 9.

The top of the insulating counterweight film frame 10 is fixed with a resistor column 19, the inner wall of the box cover 2 is embedded into an insulating sleeve 17, the inner side wall of the insulating sleeve 17 is fixed with a spring electrode 18, the spring electrode 18 is in contact fit with the resistor column 19, and the spring electrode 18 and the resistor column 19 are connected in series into a power supply circuit of a variable frequency pump 24.

Because the gas is discharged through the breathable cotton spinning film 9, there is an acting force on the breathable cotton spinning film 9, when the ventilation gas flow is large, the gas flow speed is high, so that the breathable cotton spinning film 9 is increased by the acting force, the self gravity of the insulating counterweight film frame 10 is overcome, the spring electrode 18 is moved upwards relative to the resistor column 19, the resistance of the resistor column 19 connected to a power supply circuit of the variable frequency pump 24 is increased, the split voltage of the variable frequency pump 24 is reduced, the output power of the variable frequency pump 24 is reduced, the rotating speed is reduced, and the speed of the supplied gas is reduced.

The device is provided with the spring electrode 18 and the resistor column 19, and the circuit design is skillfully utilized, so that the combination of the spring electrode 18 and the resistor column 19 is similar to the structure of a slide rheostat, the actual power of the variable frequency pump 24 and the gas exhaust speed are in a negative feedback adjusting chain, the waste of gas is prevented, and the cost is reduced.

Since the gas velocity through the air-permeable cotton spinning membrane 9 increases when the irradiation light intensity increases or the respiration increases, but when the gas supply velocity is reduced, photosynthesis or respiration is suppressed to suppress the growth of capsicum, so that the control chain is "disordered", based on which, as shown in fig. 5 to 8, 14, in the present embodiment:

the two ends of the breathable cotton spinning film 9, which are positioned at the same diameter, are provided with notches, the interiors of the notches are contacted with and fixed with film electrodes 12, the side wall of the insulating counterweight film frame 10 is provided with locking assemblies 11 in a circular array, the inner wall of the box cover 2, which is positioned at the insulating counterweight film frame 10, is fixed with a locking electromagnetic valve 13, and the film electrodes 12 are connected in series to a power supply circuit of the locking electromagnetic valve 13;

the locking assembly 11 comprises a permanent magnet 15 which is slidably connected to the radial direction of the insulating weight film frame 10 and an anti-slip pad 14 which is adhered to the side wall of the permanent magnet 15, the outer wall of the other side of the permanent magnet 15 is connected to the inner wall of the insulating weight film frame 10 through a first spring 16, and the magnetic poles of the side, opposite to the locking electromagnetic valve 13, of the permanent magnet 15 are opposite.

When the pepper embryoid is in the later stage of culture, the rhizome is in the nutrient solution, the stem and leaf are relatively photosynthetic and respiratory action, the stem and leaf are not in the nutrient solution, so the generated gas is relatively dry, but the gas supplied by the gas supply mechanism is sprayed out of the nutrient solution, the gas is relatively wet, so the air-permeable cotton spinning film 9 absorbs the moisture of the wet gas when the gas is sprayed out, the air-permeable cotton spinning film 9 is wet, the air-permeable cotton spinning film 9 evaporates after being wetted, the wetting speed of the air-permeable cotton spinning film 9 is relatively high, the moisture accumulation occurs to the air-permeable cotton spinning film 9 when the wetting speed is higher than the evaporating speed, the air-permeable cotton spinning film 9 is conductive to the membrane electrodes 12 at two ends, the locking electromagnetic valve 13 is electrified, the permanent magnet 15 is repelled by the locking electromagnetic valve 13, the spring 16 is contracted, the counter weight pad 14 is not contacted with the locking electromagnetic valve 13, the air-permeable cotton spinning film 10 can be regulated according to the negative feedback speed of the air-permeable electromagnetic valve, and the air-permeable cotton spinning film 9 is not contacted with the spring 13, and the air-permeable cotton spinning film is not contacted with the air-permeable electromagnetic valve 9, and the negative pressure is not regulated, and the air-permeable cotton spinning film 9 is not contacted with the negative-permeable electromagnetic valve is regulated, even if the negative pressure is regulated by the air-permeable electromagnetic valve is regulated.

According to the device, the locking assembly 11 is arranged, and according to the characteristics of the breathing action and photosynthesis position of the peppers, the characteristics of the contact position of the pepper culture and the nutrient solution and the characteristics of the spraying out of the inside of the nutrient solution during gas supply, the wet gas supplied by the gas can be classified with the dry gas of the photosynthesis and the breathing action, and the ionic conduction characteristics of the nutrient solution are combined, so that the negative feedback control process can accurately detect the gas supply speed, and can prevent the influence of the gas generated due to the self action of the peppers, and the control precision is increased.

In this embodiment: the transposition valve 22 can control the passage of the carbon dioxide storage tank 20 and the oxygen storage tank 21, when photosynthesis needs carbon dioxide supply, the carbon dioxide storage tank 20 can be communicated with the three-way pipe 23, the frequency conversion pump 24 is started to suck carbon dioxide into the air inlet pipe 7, when respiration needs to be used for useful oxygen supply, the transposition valve 22 is started to communicate the oxygen storage tank 21 with the three-way pipe 23, the frequency conversion pump 24 is started to suck oxygen into the air inlet pipe 7, when the transposition solenoid valve 28 is electrified, a magnetic field is generated, the magnetic fields of the two transposition solenoid valves 28 repel each other, so that the valve core I29 and the valve core II 31 repel each other to move to the left, the spring II 27 contracts, the air channel of the valve core II 31 is communicated with one air channel of the valve body 26, and when the transposition solenoid valve 28 is powered off, the valve core II 31 moves to the right under the elastic force of the spring II 27, so that the air channel of the valve core I29 is communicated with the other air channel of the valve body 26; when the valve core I29 is driven by the transposition electromagnetic valve 28, the valve core II 31 is extruded to reach the end part, at the moment, the elastic rod 33 is elastically deformed and bent outwards due to the action force between the valve core I29 and the valve core II 31, the flexible pad 32 is tightly attached to the inner wall of the valve body 26, when the valve core II 31 is driven by the spring II 27, the elastic rod 33 is elastically deformed and bent outwards due to the action force between the valve core I29 and the valve core II 31 after the valve core II 31 reaches the end part for a few days, the flexible pad 32 is tightly attached to the inner wall of the valve body 26, and gas enters the annular pipe 5 and the linear pipe 6 through the gas inlet pipe 7 and is sprayed out through the aeration holes 8, and then nutrient solution in the box body 1 is upwards moved and sprayed out to be supplied to the chillies; and because the gas is discharged through the breathable cotton spinning film 9, there is acting force on the breathable cotton spinning film 9, when the ventilation gas flow rate is high, the gas flow rate is high, so that the breathable cotton spinning film 9 is increased by acting force, so as to overcome the self gravity of the insulating counterweight film frame 10, so that the spring electrode 18 moves upwards relative to the resistance column 19, the resistance of the resistance column 19 connected to the power supply circuit of the variable frequency pump 24 is increased, the split voltage of the variable frequency pump 24 is reduced, the output power of the variable frequency pump 24 is reduced, the rotating speed is reduced, so that the gas supply speed is reduced, in addition, because the rhizome of the chilli embryoid is in the nutrient solution in the later culture period, and the stems and leaves of the chilli embryoid relatively do photosynthesis and respiration, the generated gas is relatively dry, but the gas supplied by the gas supply mechanism is sprayed from the nutrient solution, the air-permeable cotton spinning film 9 is relatively wet, so that when the air-permeable cotton spinning film 9 is sprayed out, the air-permeable cotton spinning film 9 absorbs moisture of the wet air, the air-permeable cotton spinning film 9 is wetted, the air-permeable cotton spinning film 9 evaporates after being wetted, when the air speed is too high, the air-permeable cotton spinning film 9 is relatively high, when the air speed is higher than the evaporation speed, the air-permeable cotton spinning film 9 has moisture accumulation, when the whole air-permeable cotton spinning film 9 surface is wetted, the air-permeable cotton spinning film 9 has conductivity due to ions in nutrient solution, so that the membrane electrodes 12 at two ends are conducted, the locking electromagnetic valve 13 is electrified, the locking electromagnetic valve 13 repels the permanent magnet 15, the first spring 16 contracts, the anti-slip mat 14 is not contacted with the locking electromagnetic valve 13, at the moment, the insulating counter weight film frame 10 can negatively feed back and adjust the air supply speed according to the air flow speed, otherwise, the anti-slip pad 14 is contacted with the locking electromagnetic valve 13 through the elastic force of the first spring 16, and even if the breathable cotton spinning film 9 is subjected to the action force of gas, the anti-slip pad will not move, so that negative feedback adjustment will not happen.

Example 10:

as shown in fig. 11, the following improvement is made on the basis of embodiment 9 in this embodiment, the transposition valve 22 further includes a photovoltaic plate 25, the photovoltaic plate 25 is electrically connected to the transposition solenoid valve 28, and the photovoltaic plate 25 and the sugar-free tissue culture box are in the same illumination environment.

In this embodiment, by adding the photovoltaic plate 25, the photovoltaic plate 25 can provide electric energy for the transposition electromagnetic valve 28 through the photoelectric effect, and the photovoltaic plate 25 and the sugarless tissue culture box are in the same illumination environment, so that the automatic switching of gas supply can be realized by utilizing the environment variables of photosynthesis and respiration-illumination in the later period of sugarless tissue culture.

Compared with the traditional plant propagation production process, the invention has the advantages of greatly reduced microbial contamination rate, shortened culture period, reduced rooting rate of plants and remarkably improved seedling green.

Claims

1. The pepper anther culture method is characterized by comprising the following steps of:

2. The method according to claim 1, wherein in S2, the bud picking time is 6 from No. 1 to No. 23: 00-9:30, selecting flower buds with petals equal to the calyx or purple according to 1/3 of the anther color.

3. The method for culturing pepper anther according to claim 1, wherein the minimal medium comprises major element components 4120-4940, trace element components 35.35-41.12, ferric salt 61.05-69.1, organic components 81.65-124.55, sucrose 28000-32000, agar 6800-7200;

4. A method for culturing pepper anther as claimed in claim 3, characterized in that,

the hormone component I consists of the following components in percentage by mass: KT culture medium 0.3-0.7 and NAA culture medium 2.0-3.0;

5. The method according to claim 1, wherein in the step S5, the induction medium further comprises a bacteriostatic agent comprising benzyl amoxicillin and amphotericin B, wherein the amphotericin B is water-insoluble amphotericin B.

The preparation method of the bacteriostat comprises the following steps:

6. The method according to claim 1, wherein in the step S5, the induction medium further comprises a bacteriostatic agent, the bacteriostatic agent comprising cephalosporin and amphotericin B, and the amphotericin B is water-insoluble amphotericin B.

The preparation method of the bacteriostat comprises the following steps:

7. The method according to claim 1, wherein in the step S5, the induction medium further comprises a bacteriostatic agent comprising benzyl amoxicillin, amphotericin B and cephalosporin, and the amphotericin B is water-insoluble amphotericin B.

The preparation method of the bacteriostat comprises the following steps:

8. The method for culturing pepper anther as claimed in claim 1, wherein the specific conditions in S7 are: illuminating for 14 hours each day; the illumination intensity is 2500 Lux-3000 Lux; the temperature is 23-26 ℃; the humidity is 80% -92%.

9. The pepper anther culturing method according to any one of the claims 1-8, characterized in that in the step S7, the sugar-free tissue culturing box comprises a box body (1), a supporting frame (3) fixed in the lower part of the box body (1) and a box cover (2) connected to the top of the box body (1), an aeration pipe (4) is fixed at the bottom of the box body (1), the aeration pipe (4) is formed by mutually welding and communicating a plurality of annular pipes (5) and straight pipes (6), aeration holes (8) are formed in the bottoms of the annular pipes (5) and the straight pipes (6), and the aeration pipe (4) is connected to a gas supply mechanism through an air inlet pipe (7);

the gas supply mechanism comprises a carbon dioxide storage tank (20), an oxygen storage tank (21) and a variable frequency pump (24), wherein the carbon dioxide storage tank (20) and the oxygen storage tank (21) are connected with a transposition valve (22) through a pipeline, the transposition valve (22) is connected to the gas inlet end of the variable frequency pump (24) through a three-way pipe (23), and the gas outlet end of the variable frequency pump (24) is connected to the gas inlet pipe (7) through a pipeline;

the transposition valve (22) comprises a valve body (26), and a first valve core (29) and a second valve core (31) which are connected in the valve body (26) in a sliding manner, wherein the first valve core (29) and the second valve core (31) are connected through a connecting piece (30), two parallel air passage channels are formed in the inner wall of the valve body (26), and one air passage channel is formed in each of the inner walls of the first valve core (29) and the second valve core (31); the side wall of the valve core II (31) is connected to the valve body (26) through a spring II (27), the side wall of the valve core I (29) and the inner wall of the valve body (26) are respectively fixed with a transposition electromagnetic valve (28), and the magnetic poles of the opposite sides of the two transposition electromagnetic valves (28) are opposite;

the connecting piece (30) comprises a flexible pad (32) and a plurality of circular array elastic rods (33) embedded in the flexible pad (32), wherein two sides of the flexible pad (32) and two sides of the elastic rods (33) are respectively fixed on the outer walls of one side opposite to the valve core I (29) and the valve core II (31), and the elastic rods (33) are of an arc-shaped structure bent outwards.

10. The pepper anther culturing method as claimed in claim 9, characterized in that an insulating weight film frame (10) is longitudinally and slidably connected to the inside of the box cover (2), and a breathable cotton spinning film (9) is fixed to the inside of the insulating weight film frame (10);

a resistor column (19) is fixed at the top of the insulating counterweight film frame (10), an insulating sleeve (17) is embedded into the inner wall of the box cover (2), a spring electrode (18) is fixed on the inner side wall of the insulating sleeve (17), the spring electrode (18) is in contact fit with the resistor column (19), and the spring electrode (18) and the resistor column (19) are connected in series into a power supply circuit of a variable frequency pump (24);

the breathable cotton spinning film (9) is provided with notches at two ends of the same diameter, the interiors of the notches are contacted and fixed with film electrodes (12), the side wall of the insulating counterweight film frame (10) is provided with locking assemblies (11) in a circular array, the inner wall of the box cover (2) at the insulating counterweight film frame (10) is fixed with a locking electromagnetic valve (13), and the film electrodes (12) are connected in series to a power supply circuit of the locking electromagnetic valve (13);

the locking assembly (11) comprises a permanent magnet (15) which is connected with the insulating weight film frame (10) in a sliding mode and an anti-slip pad (14) which is adhered to the side wall of the permanent magnet (15), the outer wall of the other side of the permanent magnet (15) is connected with the inner wall of the insulating weight film frame (10) through a first spring (16), and the magnetic poles of one side, opposite to the locking electromagnetic valve (13), of the permanent magnet (15) are opposite.