CN109089887B

CN109089887B - Method for tissue culture of anther

Info

Publication number: CN109089887B
Application number: CN201811254797.2A
Authority: CN
Inventors: 董亚静; 杜培粉; 缪武; 刘荣云; 杨博智; 杨石奇; 孙佳杰; 蒋翔; 李曼; 邓鑫
Original assignee: Hunan Xiangyan Seed Industry Co ltd
Current assignee: Hunan Xiangyan Seed Industry Co ltd
Priority date: 2018-10-26
Filing date: 2018-10-26
Publication date: 2021-02-26
Anticipated expiration: 2038-10-26
Also published as: CN109089887A

Abstract

The invention provides a method for tissue culture of anther, which comprises the following steps: (1) pretreatment: planting plants capable of flowering to produce said anthers in a planting shed; in the planting management process, spraying a first pesticide on the plants and/or fumigating and disinfecting the planting shed by using a second pesticide; picking flower buds when the plants enter a flowering phase; (2) anther culture: and after the buds are disinfected in an ultra-clean workbench, stripping anthers and culturing on a culture medium until embryoids are formed.

Description

Method for tissue culture of anther

Technical Field

The invention provides a method for tissue culture of anther.

Background

In the hot pepper cross breeding work, haploid breeding has the characteristics of controlling hybrid segregation, improving selection efficiency, shortening breeding period and the like, and becomes an important auxiliary breeding technology for hot pepper germplasm resource innovation.

Anther culture is the most widely applied technology for artificially inducing the parthenogernesis of crops to generate haploids. Anther culture was reported by Guha and Maheshiwarh for the first time in 1964, and a new approach for genetic breeding of higher plants was developed by culturing anther of Datura stramonium.

Although many researchers at home and abroad have seen various aspects as follows: research reports of donor genotype, hormone concentration ratio, microspore development period, addition of antibiotics and activated carbon in a culture medium and the like generally report that the problems of high pollution rate, serious anther browning, low embryoid inductivity and the like are widely existed, and the research reports are far from practical application and cannot be applied on a large scale. Although many patents and papers mention the disinfection of flower buds, the disinfection is basically similar, and almost all of them are that explants are disinfected with 75% alcohol for 30 seconds on a super clean bench, then disinfected with sodium hypochlorite or mercuric chloride for 10 to 20 minutes, and finally washed with sterile water for 3 to 4 times, but the pollution rate is still high. It is also studied to add antibiotics with a certain concentration into the culture medium to control the contamination rate, and although the contamination rate is reduced, the browning rate is increased after the antibiotics are added, the embryo yield is correspondingly reduced, even the embryoid is deformed and difficult to grow seedlings. In addition, it has been reported that the effect is not very good when silver nitrate, activated carbon, or the like is added to the medium at a low concentration.

Therefore, it is highly desirable to develop a simple and easy method for reducing the contamination rate and browning rate of anther culture and increasing the embryo induction rate.

Disclosure of Invention

The invention provides a method for tissue culture of anther, which comprises the following steps:

(1) pretreatment: planting plants capable of flowering to produce said anthers in a planting shed; in the planting management process, spraying a first pesticide on the plants and/or fumigating and disinfecting the planting shed by using a second pesticide; picking flower buds when the plants enter a flowering phase;

(2) anther culture: and after the buds are disinfected in an ultra-clean workbench, stripping anthers and culturing on a culture medium until embryoids are formed.

In one embodiment, the first pesticide is selected from a nicotinic pesticide and/or a carbamate-based fungicide.

In a particular embodiment, the first pesticide is selected from imidacloprid and/or propiconazole.

In a particular embodiment, the second pesticide is selected from isoprocarb aerosol and/or chlorothalonil aerosol.

Compared with the indoor experiment operation, the field management operation is extensive, and even greenhouse management measures are not as fine as the operation of a laboratory and also belong to the extensive category, so that the number of days between pesticide application is allowed to have a certain range difference. Thus, in a specific embodiment, the first pesticide is applied once every 10 to 20 days from the time of colonization by the plant.

In a specific embodiment, the first pesticide is applied once every 13 to 17 days from the time of plant colonization.

As above, compared with the laboratory experiment operation, the field management operation is extensive, and even the greenhouse management measures are not as fine as the laboratory operation and belong to the extensive category, so that the number of days between the application of the pesticide is allowed to be within a certain range. Thus, in a specific embodiment, the second pesticide is applied once every 5 to 10 days from the time of colonization by the plant.

In a specific embodiment, the second pesticide is applied once every 6 to 8 days from the time of plant colonization.

As above, compared with the laboratory experiment operation, the field management operation is extensive, and even the greenhouse management measures are not as fine as the laboratory operation and belong to the extensive category, so that the number of days between the application of the pesticide is allowed to be within a certain range. Thus, in one embodiment, the application of the nicotinic insecticide is from 10 to 20 grams per acre; and/or the dosage of the carbamate bactericide is 60 to 100 ml/mu.

In one embodiment, the application of the nicotinic insecticide is from 12 to 18 grams per acre; and/or the dosage of the carbamate bactericide is 75 to 85 ml/mu.

As above, compared with the laboratory experiment operation, the field management operation is extensive, and even the greenhouse management measures are not as fine as the laboratory operation and belong to the extensive category, so that the number of days between the application of the pesticide is allowed to be within a certain range. Thus, in one embodiment, the isoprocarb aerosol is fumigated on the planting shed at a rate of 200 to 300 grams per acre for 20 to 28 hours.

As above, compared with the laboratory experiment operation, the field management operation is extensive, and even the greenhouse management measures are not as fine as the laboratory operation and belong to the extensive category, so that the number of days between the application of the pesticide is allowed to be within a certain range. Thus, in one embodiment, the chlorothalonil aerosol is fumigated on the planting shed for 20 to 28 hours at a rate of 300 to 500 grams per acre.

In one embodiment, the isoprocarb aerosol fumigates the planting shed at an amount of 230 to 270 grams per acre for 22 to 26 hours.

In one embodiment, the chlorothalonil aerosol is fumigated on the planting shed at an amount of 380 to 420 grams per acre for 22 to 26 hours.

In a specific embodiment, during the planting management, the temperature in the planting shed is 21 ℃ to 30 ℃ in the daytime and 15 ℃ to 20 ℃ in the evening.

In a specific embodiment, the flower buds are four-house fighting and/or period of gypsophila 1 to 3.

As above, compared with the laboratory experiment operation, the field management operation is extensive, and even the greenhouse management measures are not as fine as the laboratory operation and belong to the extensive category, so that the number of days between the application of the pesticide is allowed to be within a certain range. Thus, in a specific embodiment, prior to sowing the seeds of the plant, the seed is treated with carbendazim (50%): the seedling substrate is stirred according to the mass ratio of (300-; and then planting the seedlings to be transplanted in the planting shed.

In a specific embodiment, the plant is seeded with carbendazim (50%): stirring the seedling substrate according to the mass ratio of (350-400) to sterilize the seedling substrate, performing plug seeding, and performing floating seedling so as to obtain seedlings to be transplanted; and then planting the seedlings to be transplanted in the planting shed.

Although the method of the invention takes pepper as an example, the method can clarify a relatively general rule, so that the method can be also expanded to anther culture of other plants and solve the same technical problems. Thus, in a specific embodiment, the plant is at least one of a Capsicum (Capsicum) plant.

In a specific embodiment, the plant is Capsicum (Capsicum annuum L.).

In one embodiment, the buds may be sterilized in step (2) with 70% to 75% alcohol and/or about 2% sodium hypochlorite, as selected according to techniques common in the art, and allowed for error.

In one embodiment, selected according to techniques common in the art, in step (2), the anthers are inoculated onto a medium and cultured in the dark at 26 to 30 ℃ (preferably 28 ℃) until embryoid bodies are formed.

In one embodiment, the medium used in step (2) may be NTH medium, selected according to techniques common in the art.

The invention has the beneficial effects that:

the method effectively reduces the pollution rate and browning rate of pepper anther culture; the method reduces or eliminates the need of adding various antibiotics, active carbon, silver nitrate and other reagents into the culture medium during anther culture, thereby saving time, material resources and manpower. And while the pollution rate and the browning rate are effectively reduced, the damage to the anther caused by antibiotics, activated carbon, silver nitrate and the like during the anther culture period is avoided, so that the embryo induction rate is improved. Wherein, the smoke fumigation of the planting shed has the most obvious effect on reducing the pollution rate and the browning rate in the anther culture. Secondly, the pesticide spraying plant has obvious effect of reducing the pollution rate and browning rate in the anther culture. Meanwhile, the flower buds in different periods also have certain influence on the browning rate and the pollution rate of the hot pepper, and the period of picking the flower buds better is determined. Finally, the environmental temperature for plant growth also has a certain influence on the browning rate and the pollution rate of the pepper, and a better plant growth temperature is determined. Among the adopted planting management measures, particularly, when the pesticide spraying plant and the smoke fumigation of the planting shed are simultaneously lifted, the pollution rate and the browning rate are greatly reduced, the embryo induction rate is obviously improved compared with that of the plant which is fumigated by using the smoke alone or fumigated by using the pesticide alone, and the synergistic effect of 1+1 > 2 is generated. In addition, other factors, such as the picking of the flower buds of 1 to 3 layers of the four-door hopper and the gypsophila paniculata, particularly the picking of the flower buds of 1 to 3 layers of the gypsophila paniculata, and the controllability of the temperature of the planting shed to control the proper environmental temperature (such as 21 to 30 ℃, particularly 26 to 30 ℃) can further improve the embryo induction rate, thereby solving the technical problem of the culture of pepper anther.

Detailed Description

The present invention will be further illustrated by the following examples of plants of the Capsicum genus (Capsicum), which are intended to be illustrative only and should not be construed as limiting the scope of the invention in any way.

The present invention will be described in detail with reference to specific examples. For each treatment of each example, 100 flower buds at the uninucleate border stage were selected for inoculation, and 500 anthers were selected.

During the development process of the pepper microspore, a tetrad period, a mononuclear early-middle period, a mononuclear border period, a binuclear period and a mature pollen grain period need to be sequentially carried out, wherein the mononuclear border period means that after the callose walls of the tetrad are completely dissolved, a central large vacuole begins to be formed, and a cell nucleus is extruded from the center to a period close to the cell wall, so that induction of an embryoid is facilitated. The morphology of the pepper flower bud is characterized in that sepals are opened, and petals slightly expose or are flush with the calyx. Since the top ends of the petals slightly expose the calyx, the petals of the pepper are thin, and the anther infection by external bacteria is difficult to resist.

The pepper (Capsicum annuum L.) is RX 15-495-1.

The substrate for seedling culture is purchased from Shandong Xiangdao Biotech Co.

Carbendazim was purchased from Yifeng pesticide, Inc., Anyang.

Isoprocarb aerosol with 15% active ingredient was purchased from vernal agrichemical ltd, south of Henan.

Chlorothalonil aerosol with 10% of active ingredient was purchased from fine chemical industries, ltd.

Potassium sulfate type compound fertilizers are available from stanli chemical fertilizer, ltd.

Imidacloprid was purchased from Zhejiang Hai Zhejiang Utility chemical Co.

Pulikes with 72.2% active ingredient were purchased from zhongnong lihua (tianjin) agrochemicals limited.

The formula of the induction culture medium for anther culture comprises the following components:

NTH basic culture medium + 0.2mg/L NAA + 1.0mg/L KT + 3wt% sucrose + 0.8wt% agar. Wherein, the formula of the NTH minimal medium is as follows: 720mg/L NH₄NO₃950mg/L KNO₃166mg/L of CaCl₂185mg/L MgSO₄·7H₂O, 68mg/L KH₂PO₄10mg/L of H₃BO₃25mg/L of MnSO₄·4H₂O, 10mg/L ZnSO₄·7H₂O, 0.25mg/L MoO₃0.025mg/L of CoCl₂·6H₂O, 27.8mg/L FeSO₄·7H₂O, 37.8mg/L of Na₂·EDTA·2H₂O, 100mg/L inositol, 5mg/L nicotinic acid, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L thiamine hydrochloride, 2mg/L glycine, 0.5mg/L folic acid and 0.05mg/L biotin.

Sterilizing at pH of 5.8-6.0 and 121 deg.C under 103.4KPa for 20 min.

Example 1

(1) Seedling culture: before sowing, the seed is mixed with carbendazim (50%): and (3) sterilizing the seedling culture substrate by stirring the seedling culture substrate in a mass ratio of 1: 375. And (5) plug seeding and floating seedling.

(2) Cultivation management: and (2) planting the seedlings obtained in the step (1) in a greenhouse with controllable temperature, wherein the planting density is 40 x 40 cm, the temperature of the greenhouse is controlled to be 26-30 ℃ in the daytime, 15-20 ℃ at night, and the seedlings are isolated by a gauze. Watering by drip irrigation every 10 days from the beginning of field planting, and watering the potassium sulfate compound fertilizer at the same time. The imidacloprid and pulicarb are sprayed on the plants at 15-day intervals. The dosage of imidacloprid is 15 g/mu (15 g imidacloprid is dissolved in 45L of water), and the dosage of puleg is 75 ml/mu (75 ml puleg is dissolved in 45L of water).

(3) Picking flower buds: in the early flowering period and full flowering period after the plant blooms to 4 blossoms, 8: 00 to 10: 00, picking up robust plants, wherein the external morphological characteristics of the robust plants are that the petals are equal to the calyx (namely sepals: 1), most of the flower buds are in the single-core side-by-side period during the microspore development period, putting the flower buds into an ice box by using a sealing bag, putting the ice box back to a laboratory, and putting the ice box in a 4-degree refrigerator for 48 hours for treatment.

The flower picking parts are respectively as follows: four-door fights, 1-3 layers of babysbreath and 4 layers or more of babysbreath.

(4) And (3) disinfection: on an ultraclean bench, the bud (explant) surface was sterilized with 70% by volume alcohol for 30 seconds, then with 2% by volume sodium hypochlorite for 10 to 12 minutes, and then rinsed 3 times with sterile water.

(5) Culturing: the anther is stripped from the flower bud by using tweezers on a clean bench, the filaments are thoroughly removed (the anther is prevented from being damaged as much as possible in the operation), the anther is inoculated into an induction culture medium, 20 anthers/dish (the diameter of the culture dish is 60mm) is covered, and the culture dish is covered and sealed by using a Parafilm membrane. The culture dish inoculated with the anther is arranged in a small box and cultured in the dark at the temperature of 28 ℃ until embryoid appears. Each treatment was inoculated with 500 anthers, repeated three times. Statistical analysis was performed using SPSS.

The browning rate is the number of browned anthers/inoculated anthers × 100%, and is counted after 8 days of culture, and the average of three replicates is taken as the average browning rate.

The contamination rate is the number of contaminated anthers/the number of inoculated anthers × 100%, and counted after 15 days of culture, and the average contamination rate was calculated as the average of three replicates.

The embryo emergence rate is the number of anthers producing embryoid/the number of inoculated anthers × 100%, and is counted after 40 days of culture, and the average embryo emergence rate is the average of three replicates.

The results are shown in Table 1.

Example 2

The temperature of the greenhouse is 16-20 ℃ in the day and 15-20 ℃ at night. The other operations were the same as in example 1.

The results are shown in Table 1.

Example 3

The temperature of the greenhouse is 21-25 ℃ in the day and 15-20 ℃ at night. The other operations were the same as in example 1.

The results are shown in Table 1.

Example 4

The temperature of the greenhouse is 31-35 ℃ in the day and 15-20 ℃ at night. The other operations were the same as in example 1.

The results are shown in Table 1.

Example 5

No chemical spraying is carried out on the plants. After planting, the greenhouse is fumigated for 24 hours by isoprocarb aerosol with the dosage of 250 g/mu in a greenhouse. The other operations were the same as in example 1.

The results are shown in Table 1.

Example 6

No chemical spraying is carried out on the plants. After planting, the greenhouse is fumigated for 24 hours by using chlorothalonil aerosol with the dosage of 400 g/mu every other week. The other operations were the same as in example 1.

The results are shown in Table 1.

Example 7

After planting, the greenhouse is fumigated for 24 hours by isoprocarb aerosol with the dosage of 250 g/mu in a greenhouse. The other operations were the same as in example 1.

The results are shown in Table 1.

Example 8

No chemical spraying is carried out on the plants. The other operations were the same as in example 1.

The results are shown in Table 1.

Comparative example 1

(2) Cultivation management: and (2) planting the seedlings obtained in the step (1) in open field at a planting density of 40 x 40 cm, watering the seedlings and a potassium sulfate compound fertilizer every 10 days, and spraying imidacloprid and pulex to the seedlings at the same time every 15 days.

The other operations were the same as in example 1.

The results are shown in Table 1.

Comparative example 2

0.25% activated carbon and 6mg/L silver nitrate were added to NTH medium, as in comparative example 1.

The results are shown in Table 1.

Comparative example 3

20mg/L streptomycin sulfate and 20mg/L cephamycin are added into the NTH culture medium. The rest is the same as in comparative example 1.

The results are shown in Table 1.

TABLE 1 statistics of anther culture results at flowering phase in four-door fill period

Examples	Average browning rate of anther (%)	Average contamination ratio of anther (%)	Average embryo rate (%)
				Example 1	17.4Bb	12.5Dd	5.9Bb
Example 2	18.8Bb	16.5Dd	3.3Dd
				Example 3	17.7Bb	15.2Dd	4.6Cc
Example 4	36.8Aa	32.9Bb	1.0Ee
				Example 5	9.6Cc	10.8Dd	6.3Bb
Example 6	10.1Cc	11.5Dd	5.7Cc
				Example 7	8.6Cc	9.2Ee	9.8Aa
Example 8	21.2Bb	28.5Cc	0.5Ff
				Comparative example 1	36.1Aa	38.7Aa	1.2Ee
Comparative example 2	20.6Bb	39.1Aa	2.0Ee
				Comparative example 3	38.4Aa	27.6Cc	1.7Ee

TABLE 2 statistics of anther culture results at the blooming period of the 1-3 layers of gypsophila

Examples	Average browning rate of anther (%)	Average contamination ratio of anther (%)	Average embryo rate (%)
				Example 1	15.8Dd	12.8Dd	6.8Bb
Example 2	16.5Dd	15.4Dd	3.7Dd
				Example 3	16.1Dd	13.6Dd	5.9Cc
Example 4	31.3Cc	36.7Bb	1.2Ff
				Example 5	10.2Ee	8.5Ee	7.2Bb
Example 6	10.8Ee	9.3Ee	6.9Bb
				Example 7	7.3Ff	8.5Ee	12.9Aa
Example 8	36.3Bb	45.6Aa	1.0Ff
				Comparative example 1	43.8Aa	49.1Aa	1.6Ff
Comparative example 2	18.3Dd	48.6Aa	2.5Ee
				Comparative example 3	41.0Aa	24.3Cc	2.3Ee

TABLE 3 statistics of anther culture results in blooming period of 4 or more layers of Gypsophila paniculata

Examples	Average browning rate of anther (%)	Average contamination ratio of anther (%)	Average embryo rate (%)
				Example 1	26.6Dd	20.5Dd	3.7Bb
Example 2	33.8Cc	17.6Ee	2.3Cc
				Example 3	26.4Dd	22.3Dd	3.7Bb
Example 4	43.7Bb	41.8Bb	0.3Ff
				Example 5	20.3Ee	14.1Ff	4.1Bb
Example 6	20.9Ee	17.1Ee	3.9Bb
				Example 7	18.9Ee	13.6Ff	6.3Aa
Example 8	57.6Aa	68.9Aa	0.4Ff
				Comparative example 1	55.6Aa	70.2Aa	0.7D
Comparative example 2	32.7Cc	68.5Aa	1.3Dd
				Comparative example 3	55.9Aa	32.5Cc	0.9Ee

Note: in the table, capital letters represent extremely significant differences (P <0.01), and lowercase letters represent significant differences (P <0.05)

Analysis of results

As can be seen from Table 1, in examples 1, 2 and 3, the greenhouse temperature is 16-30 ℃ in the daytime, and the browning rate and the pollution rate of the anthers have no obvious difference in the initial flowering period of flower picking; when the temperature is over-high at 30-35 ℃, the browning rate and the pollution rate of anther culture are higher, the embryo induction rate is correspondingly reduced, and the browning rate and the pollution rate of example 4 are obviously increased, so that the differences between examples 1-3 and example 4 are the most obvious, but the overall effects of examples 1 and 3 are better than that of example 2; the anthers of examples 1, 2, 3 and 4 all have significant differences in embryo-out rate, wherein the embryo-out rate is higher for example 1 and example 3, and lower for example 2. The difference of the anther browning rate among the examples 5, 6 and 7 is not large, and the difference of the pollution rate of the example 7 and the examples 5 and 6 is obvious; the browning rate of smoke fumigation in examples 5, 6 and 7 and the browning rate of the pesticide sprayed in example 1 are obviously reduced, the embryo yield of example 7 is obviously increased compared with that of example 1, and the difference between the browning rate and the pollution rate of example 8 and examples 5, 6 and 7 is obviously increased; the browning rate and the pollution rate between examples 5, 6, 7 and 8 and comparative example 1 are obviously reduced; compared with the effects of adding antibiotics and anti-browning agents into the culture medium in the comparison examples 2 and 3, the fumigation effects of the medicaments in the examples 5, 6 and 7 are obvious, and the browning rate and the pollution rate are obviously reduced; the browning rate and the pollution rate of the embodiment 5 and the embodiment 6 are not obviously different, and the effect of the embodiment 5 on the embryo yield of the embodiment 6 on the fumigation with the isoprocarb smoke is slightly better than that of the embodiment 6 on the fumigation with the chlorothalonil smoke. From the anther culture in the whole four-door period, the fumigation and the pesticide spraying in the embodiment 7 are carried out simultaneously, the embryo emergence rate is the highest, and the browning rate and the pollution rate are the lowest.

As shown in table 2, the browning rate and the contamination rate of the anther culture of the gypsophila paniculata in the 1-3 layers of the florescence are not obviously different among the examples 1, 2 and 3, but are obviously different from the example 4, namely the example 4 is obviously improved; the anther embryo-out rates of examples 1, 2, 3 and 4 are all obviously different, wherein the embryo-out rate of example 1 is the highest, and the embryo-out rate of example 3 is good. The browning rate and the pollution rate of the examples 5 and 6 are obviously reduced compared with the example 1, the embryo emergence rate difference is not obvious, and the data show that the effect of single smoke fumigation is slightly higher than that of single pesticide spraying; the differences of the browning rate, the pollution rate and the embryo yield between the example 5 and the example 6 are small, while the browning rate of the example 7 is obviously reduced and the difference of the pollution rate is small; compared with the fumigation effects of the medicaments added into the culture media in the comparative examples 2 and 3, the fumigation effects of the medicaments in the examples 5, 6 and 7 are obvious, the browning rate and the pollution rate are obviously reduced, and the embryo yield is obviously increased; from the anther culture of the gypsophila paniculata in the flowering period of 1 to 3 layers, the highest embryo emergence rate is observed by simultaneously carrying out fumigation and pesticide spraying in example 7, then smoke fumigation alone in examples 5 and 6, then pesticide spraying alone in example 1, and treatment without fumigation and pesticide spraying in example 8 is the lowest.

Table 3 shows that the greenhouse temperature control in the daytime of example 1 and example 3 at 21 ℃ -30 ℃ has no significant difference, the greenhouse temperature control in the daytime of example 1 is below 20 ℃ (as in example 2) and above 30 ℃ (as in example 4), the browning rate of example 2 and example 4 is significantly higher than that of example 1, and the pollution rate of example 2 is lower than that of example 1 after the greenhouse temperature in the daytime is lower than 20 ℃; by the later stage of the flowering period, compared with examples 5, 6 and 7, the browning rate and the pollution rate of the non-fumigation and non-spraying agent treatment in example 8 are obviously increased, and the embryo emergence rate is obviously reduced. Compared with the effect of adding antibiotics and anti-browning agents into the culture medium in the comparison of the proportion 2 and the proportion 3, the fumigation effect of the medicaments in the examples 5, 6 and 7 is more obvious, the browning rate and the pollution rate are obviously reduced, and the embryo yield is obviously increased. The fumigation and the spraying of the medicament are carried out simultaneously in the embodiment 7, so that the browning rate and the pollution rate are lowest, and the embryo emergence rate is highest.

The results in tables 1-3 show that the above-mentioned two steps of example 7, fumigation in greenhouse for 24 hours with isoprocarb aerosol every other week and at the dosage of 250 g/mu, and simultaneous plant spraying with imidacloprid and pulex every 15 days, result in the lowest browning rate and contamination rate of anther culture and the highest embryo yield; the anther culture example 7, which is the most suitable for the florescence of 1-3 layers of gypsophila, has the highest embryo yield of 12.9%, the next four-door period of 9.8%, and the last 6.3% of the florescence of 4 layers or more of gypsophila.

Claims

1. A method of tissue culture of anthers comprising the steps of:

(1) pretreatment: planting plants capable of flowering to produce said anthers in a planting shed; spraying a first pesticide on the plants and fumigating and disinfecting the planting shed by using a second pesticide in the planting management process; picking flower buds when the plants bloom to the initial flowering period and the full flowering period after 4 douches, wherein the flower picking parts are 1-3 layers of gypsophila;

in the planting management process, the temperature in the planting shed in the daytime is 26-30 ℃, and the temperature in the evening is 15-20 ℃;

the first pesticide is selected from imidacloprid and propiconazole;

the second pesticide is selected from isoprocarb aerosol and/or chlorothalonil aerosol;

the plant is pepper; (2) anther culture: after the flower bud is disinfected in an ultraclean workbench, the anther is stripped and cultured on a culture medium in the dark at 28 ℃ until an embryoid is formed, wherein the formula of the culture medium is NTH basic culture medium plus 0.2mg/L NAA plus 1.0mg/L KT plus 3wt% sucrose plus 0.8wt% agar.

2. The method of claim 1, wherein the first pesticide is applied once every 10 to 20 days from the time of plant colonization;

applying the second pesticide once every 5 to 10 days from the time of plant colonization.

3. The method of claim 2, wherein the first pesticide is applied every 13 to 17 days and the second pesticide is applied every 6 to 8 days from the time of plant colonization.

4. The method according to claim 1, wherein the imidacloprid is used in an amount of 10 to 20 grams/acre; the dosage of the pulex is 60 to 100 ml/mu.

5. The method according to claim 4, wherein the imidacloprid is used in an amount of 12 to 18 grams/acre; the dosage of the pulex is 75 to 85 ml/mu.

6. The method of claim 1 wherein the isoprocarb aerosol is fumigated on the planting shed at a rate of 200 to 300 grams per acre for 20 to 28 hours; and/or the chlorothalonil aerosol fumigates the planting shed for 20 to 28 hours at a rate of 300 to 500 grams per acre.

7. The method of claim 6 wherein the isoprocarb aerosol is fumigated at a rate of 230 to 270 grams per acre for 22 to 26 hours on the planting shed; and/or the chlorothalonil aerosol fumigates the planting shed for 22 to 26 hours at an amount of 380 to 420 grams per acre.

8. The method according to any one of claims 1 to 7, wherein, prior to sowing the seeds of the plant, the plant is inoculated with a mixture of 50% carbendazim: the seedling substrate =1 (300-450) is stirred according to the mass ratio to be disinfected, sowed in a plug tray and cultured in a floating manner, so that the seedlings to be transplanted are obtained; and then planting the seedlings to be transplanted in the planting shed.

9. The method as claimed in claim 8, wherein, before sowing the seeds of the plant, the plant is sown with a mixture of 50% carbendazim: the seedling substrate =1 (350-400) is stirred according to the mass ratio to be disinfected, sowed in a plug tray and cultured in a floating manner, so that the seedlings to be transplanted are obtained; and then planting the seedlings to be transplanted in the planting shed.