Method for improving pepper anther tissue culture embryoid induction rate and reducing pollution rate

Abstract

The invention relates to the technical field of pepper anther tissue culture, and particularly discloses a method for improving pepper anther tissue culture embryoid induction rate and reducing pollution rate, and the method is characterized in that a culture medium containing GABA (gamma-aminobutyric acid) is adopted for culture. Specifically, the culture medium comprises an induction culture medium for inducing embryoid formation, an embryoid culture medium for culturing embryoid seedlings and a seedling growth culture medium for culturing seedlings to be mature. On the basis of a conventionally used culture medium formula, 50 mM of GABA is added into the culture medium in each stage, so that the embryoid induction rate can be remarkably improved, the pollution rate can be reduced, and finally the purpose of improving the culture success rate of the pepper anther is achieved. In the anther tissue culture process, compared with a conventional culture medium, the pollution rate of the culture medium added with 50 mM of GABA is reduced by 4.5-2.5 times, the embryoid induction rate is averagely improved by 1.6-2.2 times, and the survival haploid seedling is improved by 1.6-3.3 times.

Description

Technical Field

[0001] The present invention relates to the technical field of pepper anther tissue culture, and specifically to a method for improving the induction rate of embryoids in pepper anther tissue culture and reducing the contamination rate. Background Art

[0003] Pepper breeding methods include traditional breeding (selection breeding, cross breeding, backcross breeding), modern biotechnology breeding (molecular marker-assisted selection, genetic engineering, cell engineering), mutagenesis breeding, polyploid breeding, genomics-assisted breeding, and phenomics-assisted breeding, etc. These methods can be used in combination to efficiently cultivate disease-resistant, stress-resistant, high-yield, and high-quality pepper varieties. Among them, anther culture haploid breeding occupies an important position in pepper breeding. Anther culture haploid breeding can quickly obtain homozygous lines and significantly shorten the breeding cycle by inducing microspores or pollen to form haploid plants and doubling the chromosomes. This technology improves the selection efficiency, facilitates the fixation of target traits and the screening of recessive excellent genes, and at the same time promotes genetic improvement and germplasm innovation, enriching genetic diversity. Since anther culture haploid breeding has significant advantages in disease-resistant breeding, quality improvement, etc., and can efficiently and accurately cultivate disease-resistant, high-quality, and high-yield pepper varieties, anther culture is an indispensable key technical means in the current pepper breeding field.

[0004] Anther culture has great potential in the rapid propagation and genetic improvement of peppers. By inducing haploid plants, anther culture can obtain completely homozygous inbred lines within a single generation, significantly shortening the inbreeding generations required for traditional breeding. This method not only reduces production costs but also greatly improves the propagation efficiency. In addition, as a precise breeding tool, anther culture helps to cultivate new pepper varieties with stronger resistance or adaptability while maintaining genetic stability. These advantages are of great significance for the efficient breeding and genetic improvement of peppers. The most important link in anther culture is the formation of embryoids, which directly determines the successful induction of haploid plants. To overcome this key link, the following aspects can be considered: in terms of material selection, anthers at the late uninucleate to early binucleate stage should be selected, and genotypes that are easy to induce or those with improved induction rates through genetic improvement should be prioritized; in the culture system, the ratio of hormones, carbon sources, and additives in the medium needs to be finely optimized; in terms of environmental regulation, temperature, light, and humidity should be precisely controlled; in addition, the dedifferentiation ability of anthers can be activated through low-temperature or chemical treatment during the pretreatment stage. Moreover, when embryoids germinate, they should be promptly transferred to a suitable medium to maintain a stable environment to ensure the smooth regeneration of plants. In anther culture, there are two ways to induce the formation of embryoids. One is to first induce pollen to form callus, which is an undifferentiated cell mass, and then embryoids are differentiated from the callus. The other is to directly induce embryoids. Direct induction of embryoids has many advantages. It can reduce the number of cell divisions, lower the risk of variation, and better maintain genetic stability; it does not need to go through the callus stage, greatly shortening the culture cycle and resulting in faster seedling formation; moreover, the embryoids formed by direct induction have better physiological activity, the developed plants have strong growth potential, and the probability of producing abnormal seedlings is low, which can increase the output rate of effective seedlings.

[0005] Since the first successful induction of haploid embryos from pepper anthers by previous researchers, significant progress has been made in anther culture technology for pepper breeding. However, its practical application still faces many challenges, including the influence of factors such as genotype dependence of donor plants, microspore development stage, pretreatment methods, and culture conditions. Among them, genotype dependence and the high contamination rate during the culture process are still the main obstacles restricting the success of pepper anther culture.

[0006] Contamination is a common problem in plant tissue culture. During rapid propagation, contamination usually stems from incomplete sterilization of the culture medium, inoculation tools, laminar flow hood, or inoculation room, as well as improper aseptic techniques during the operation process. In addition, the surface of plant materials exposed to the environment for a long time may be attached with microorganisms, leading to exogenous contamination. Some microorganisms can even invade the intercellular spaces or internal cells of plant tissues, and these microorganisms cannot be completely eliminated by surface sterilization, thus causing endogenous contamination. Once introduced into the culture system, it will seriously affect the culture effect. These contaminants not only reduce the success rate of tissue culture but also increase the production cost. To cope with exogenous contamination, disinfectants such as ethanol and sodium hypochlorite are usually used. These methods can effectively kill pathogens, but the concentration of disinfectants needs to be strictly controlled to avoid poisoning or damaging plant tissues.

[0007] In the existing anther culture technology, the induction rate of embryoids is low, and the induction rate of callus is higher than that of embryoids. The contamination rate is high, which affects the formation of embryoids and callus in anther tissue culture. Summary of the Invention

[0008] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for improving the anther tissue culture of peppers, so as to at least achieve the effects of increasing the induction rate of embryoids, reducing the contamination rate, and ultimately improving the success rate of pepper anther culture.

[0009] The purpose of the present invention is achieved through the following technical solutions: A method for reducing the contamination rate of pepper anther tissue culture: Culturing with a culture medium containing GABA (γ-aminobutyric acid).

[0010] In some embodiments, the culture medium includes: an induction medium for inducing the formation of embryoids; an embryoid medium for cultivating embryoids into seedlings; and a seedling growth medium for cultivating seedlings to maturity.

[0011] In some examples, the addition amount of GABA in the induction medium, embryoid medium, and seedling growth medium is 50 mM.

[0012] In some other embodiments, the method includes the following steps: S1: Inoculate pepper anthers on the induction medium for culture until embryoids grow on the pepper anthers to obtain embryoids; the induction medium includes MS medium, sucrose, activated carbon, agar powder, silver nitrate, 2,4-D, 6-BA, and GABA; S2: Transfer the embryoids to the embryoid medium for culture until the embryoids develop young roots and young leaves to obtain seedling embryoids; the embryoid medium includes MS medium, sucrose, agar powder, silver nitrate, 2,4-D, 6-BA, and GABA; S3: Transfer the formed embryoids to a seedling growth medium for cultivation to obtain regenerated plants; The seedling growth medium includes MS medium, sucrose, agar powder, IBA, and GABA.

[0013] In some examples, the method of culturing pepper anthers in step S1 is specifically: perform dark heat shock treatment at 33 - 36 °C for 6 - 8 days, then perform dark culture at 25 - 28 °C for 20 - 30 days, and finally perform light culture until embryoids grow from the anthers.

[0014] In some examples, by weight, the induction medium includes 4.43 parts of MS medium, 30 parts of sucrose, 0.4 part of activated carbon, 7 parts of agar powder, and 10.3 parts of GABA; The induction medium further includes: 4 mg / L silver nitrate, 0.5 mg / L 2,4 - D, and 0.5 mg / L 6 - BA.

[0015] In some examples, by weight, the embryoid medium includes 4.43 parts of MS medium, 30 parts of sucrose, 7 parts of agar powder, and 5.15 parts of GABA; The embryoid medium further includes: 4 mg / L silver nitrate, 0.5 mg / L 2,4 - D, and 0.5 mg / L 6 - BA.

[0016] In some examples, by weight, the seedling growth medium includes 4.43 parts of MS medium, 30 parts of sucrose, 7 parts of agar powder, and 5.15 parts of GABA; The seedling growth medium further includes 0.1 mg / L IBA.

[0017] In some examples, the method for obtaining pepper anthers is: In the initial stage of pepper flowering, add a fertilizer containing GABA for cultivation, then pick the flower buds, sterilize them with alcohol, and then take out the pepper anthers from the flower buds.

[0018] The beneficial effects of the present invention are: Based on the conventionally used culture medium formula, 50 mM GABA is added to the culture media at each stage in the present invention, which can not only significantly improve the embryoid induction rate but also reduce the contamination rate, ultimately achieving the purpose of improving the success rate of pepper anther culture. Among them, during the anther tissue culture process, adding 50 mM GABA to the culture medium reduces the contamination rate by 4.5 - 2.5 times compared with the conventional culture medium, the embryoid induction rate is increased by an average of 1.6 - 2.2 times, and the surviving haploid seedlings are increased by 1.6 - 3.3 times. Description of the Drawings

[0019] Figure 1 Calculation results of the contamination rate, seedling formation rate, browning rate, and induction rate for Example 1 and Comparative Examples 1-4 in Experimental Example 1; Figure 2 Photos of each stage of tissue culture of the middle section of hot pepper in Example 1; in the figure, A is the anther taken out from the flower bud at the beginning of the culture in step 3); B is the swelling of the anther during the induction culture; C is the emergence of the embryoid from the anther during the induction culture; D-F are different developmental stages of the embryoid, D is the elongated embryoid, E is the embryoid turning green, F is the cotyledon-type embryo; G is the embryoid transplanted onto the embryoid medium; H is the gradual development of the roots of the embryoid to form a regenerated plant with roots, stems, and leaves; 1 is the tissue culture seedling with 6 to 8 true leaves; J is the plant developed after growing in the soil for 2 weeks; K is the browning anther with abnormal anther development and gradually turning brown; L is the embryo forming callus and unable to continue normal development. Detailed implementation manners

[0020] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings, but the protection scope of the present invention is not limited to the following description.

[0021] Sources of the antibacterial agents used in the following examples and comparative examples: Plant broad-spectrum antibiotic ppm (PPMTM, Plant Preservative Mixture, is a broad-spectrum antifungal agent), brand: Ximeijie; BABA (β-aminobutyric acid / DL-3-aminobutyric acid chemical formula: C4H9NO2 CAS number: 541-48-0 product number: A44207-5G), purchased from Sigma-Aldrich, USA; GABA (γ-aminobutyric acid chemical formula: C4H9NO2 CAS number: 56-12-2 product number A800350-100g), purchased from Sigma-Aldrich, USA; penicillin (CAS number: 61-33-6 chemical formula: C 16 H 18 N2O4S A800090-5g), purchased from Beijing Coupling.

[0022] The hot pepper variety used: B23-3, linear pepper, scientific name Capsicum annuum L, the seed source comes from Jiangsu Surun, the plant height is 88-90 cm, the plant width is 86-88 cm, the fruit is green and long and linear, the fruit setting is concentrated, and it is early maturing. The green fruit is green, the red fruit is bright red, the fruit shape index (the ratio of the longitudinal diameter to the transverse diameter of the fruit) is 18.3-24.4, the capsaicin content is 1.8-2.2 mg / g, and the crude fat content is 0.85-0.92 mg / g.

[0023] Example 1 Perform tissue culture on chili anthers, and add GABA as a plant resistance inducer to the induction medium, embryoid medium, and seedling growth medium. The specific method is as follows: 1) Chili cultivation management and flower bud treatment: Carry out plug seedling of chili variety B23-3 in the plant culture room in January 2023; The seedlings are planted in a soilless cultivation mode of foam planting trough + non-woven fabric + nutrient substrate in a plastic greenhouse. Lay a 288-hole water-permeable plug tray at the bottom of the foam planting trough to control water; Lay a layer of non-woven fabric with good water permeability, firm and durable on it; Fill the substrate above the non-woven fabric. Before filling, mix perlite and ternary compound fertilizer into the substrate according to a volume ratio of 1:3, and the depth of the substrate soil layer is 35-40 cm. The water and fertilizer management adopts drip irrigation combined with a proportional pump, and the drip irrigation EC value of the water-soluble fertilizer (product of Chengdu Institute) is controlled at 1.5-2.5 mS / cm. When planting, the seedlings need to be planted near the drip irrigation holes to ensure sufficient water absorption. At the initial stage of flower appearance at the end of March, add 0.2-0.5% of a certain proportion of GABA (CAS No.: 56-12-2 A800350-100g) to the water-soluble fertilizer. Start picking flower buds at the end of April, and the picking times are from 7 to 8 am from April to May and from 6 to 7 am from May to June. The flower buds are required to have a petal to sepal ratio of about 1, a flower bud length of 2-3 mm, and 1 / 3 of the anthers being purple. Pick the flower buds according to the material numbers, pack them well in a plastic-sealed bag, and bring them back to the laboratory in an ice box. 2) Flower bud sterilization: Perform the sterilization and inoculation operations of the flower buds on the super clean bench in the tissue culture room (note: the ultraviolet lamp of the super clean bench has been turned on in advance for 30 minutes of disinfection), and the flower bud treatment process is carried out on ice. Flower bud stripping: Prepare a sterilized stainless steel plate, lay sterilized filter paper in the plate, and place ice cubes at the bottom of the plate. Take out the flower buds in the plastic-sealed bag, gently remove the outer sepals with tweezers, and put them into a sterilized tissue culture bottle to prepare for sterilization. Flower bud sterilization: Disinfect the stripped flower buds with an appropriate amount of 75% alcohol (submerging the flower buds) for 30 seconds and shake. Take out the supernatant, add 0.1% mercuric chloride, and add 1 drop of Tween 80 (15 drops in 100 ml of solution), shake for 7 minutes, and finally wash with sterilized water multiple times until there are no bubbles to obtain sterilized chili flower buds.

[0024] 3) Anther inoculation and heat shock treatment: Carefully remove the anthers from the sterilized flower buds using sharp tweezers and inoculate them into the prepared induction medium in a petri dish. Inoculate 20 - 30 anthers per dish, and then seal it with parafilm in two layers. Place the inoculated petri dish in a 35°C constant temperature incubator for a one - week dark heat shock treatment. Then transfer the petri dish to a dark culture room at 25 - 28°C and culture for 20 - 30 days. Observe and record the occurrence of embryoids every 7 days during this period. Then conduct light culture. Place the petri dish in a light culture room. Use a T8 plant growth lamp as the light source, with a light intensity of 3500 - 5000 LUX, long - day (photoperiod: 16 h light, 8 h dark); the humidity is between 50% - 70%. When embryoids grow on the inoculated anthers, transfer them to a petri dish with embryoid medium to continue growing, with 5 - 15 embryoids per dish. At this time, the light intensity should be ≥5500; after developing young roots and young leaves, transfer them to a culture bottle with seedling growth medium to continue growing (light culture), with 1 seedling per dish. The formula of the medium is as follows: Induction medium: Embryoid (development) medium: Seedling growth medium: 4) Hardening off of tissue - cultured seedlings and soil cultivation management: Backup and hardening off of tissue - cultured seedlings: To ensure the survival rate of seedlings and subsequent identification work, it is necessary to backup the seedlings. When the seedlings grow to 5 - 8 cm in length, open the bottle cap on the ultra - clean workbench, cut off the apical bud 1.5 - 2.5 cm from the top of the seedling, and remove the leaves, only retaining the apical meristem and the stem. Inoculate the treated apical bud into a new seedling growth medium for light culture and change the medium monthly. When both the original tissue - cultured seedlings and the backup seedlings grow new leaves and new roots and become seedlings, they can be hardened off.

[0025] During hardening off, to adapt to the micro - environment of the culture room, first loosen the bottle cap of the original tissue - cultured seedlings to allow environmental air to enter the bottle and acclimatize for 7 - 10 days. Then, open the bottle cap and add an appropriate amount of pure water to the bottle, and adapt for 2 - 7 days until the seedlings are healthy and well - adapted, and then proceed with transplantation.

[0026] 5) Soil culture transplantation and management: First, remove the culture medium carried by the tissue-cultured seedlings after hardening off. Gently rinse the roots of the tissue-cultured seedlings, and avoid damaging the roots during the operation. Rinse them clean with warm water at 25-35 °C. Then, soak the rinsed tissue-cultured seedlings for 10-20 minutes in a mixed solution containing carbendazim (800-1000 times dilution) and rooting powder (20-40 mg / kg), and then carry out soil culture transplantation. After transplantation, cover with a film or plastic bag to keep the humidity at 80-95%. Add perlite (1:3) to the soil culture substrate, and supplement the substrate with MS nutrient solution. After 10-15 days of acclimatization, the covering film can be removed, and then enter the daily management.

[0027] Comparative Example 1 Carry out pepper anther tissue culture. The method is the same as that in Example 1 and is carried out simultaneously with Example 1. The difference is that two groups of experiments are carried out simultaneously, and ppm with addition amounts of 0.0625% and 0.075% are added as plant resistance inducers to the induction medium, embryoid medium, and seedling growth medium (the addition amounts here are the conventional addition amounts recommended by the reagent manufacturer).

[0028] Comparative Example 2 Carry out pepper anther tissue culture. The method is the same as that in Example 1 and is carried out simultaneously with Example 1. The difference is that two groups of experiments are carried out simultaneously, and BABA with addition amounts of 25 mM and 50 mM are added as plant resistance inducers to the induction medium, embryoid medium, and seedling growth medium.

[0029] Comparative Example 3 Carry out pepper anther tissue culture. The method is the same as that in Example 1 and is carried out simultaneously with Example 1. The difference is that two groups of experiments are carried out simultaneously, and penicillin with concentrations of 2.50% and 5.00% (accounting for the whole medium) are added as plant resistance inducers to the induction medium, embryoid medium, and seedling growth medium (the addition amounts here are the conventional addition amounts recommended by the reagent manufacturer).

[0030] Experimental Example 1 Count the number of induced embryos, inoculated anthers, seedlings, browned anthers, inoculated culture dishes, and contaminated culture dishes during the cultivation processes of Example 1 and Comparative Examples 1-3: Among them, the number of induced embryos: Under specific culture medium formulations and culture conditions, the number of anthers that successfully induce the formation of typical embryoids through anther in vitro culture. It is counted in units of individual anthers. When a single embryoid is produced from the same anther or multiple embryoids are produced from the same anther, it is counted as 1 effective induction number.

[0031] Number of inoculated anthers: The total number of effective anthers that are separated from donor plants at the initial stage of the experiment, surface-sterilized, and completely inoculated into the culture medium for in vitro culture. It does not include ineffective inoculated individuals with structural damage caused by operation damage.

[0032] Number of seedlings: The number of regenerated plant individuals that complete the differentiation of complete plants through the somatic embryo regeneration pathway and have standard morphological characteristics for transplanting and survival (at least having roots and more than 3 true leaves). Abnormal development individuals such as deformed seedlings and vitrified seedlings need to be excluded.

[0033] Number of browned anthers: The number of anthers in which the culture medium and the anthers themselves turn significantly brown due to the oxidation of phenolic substances during the culture process, and accompanied by the loss of cell activity and the loss of regeneration potential. Local browning samples caused by mechanical damage need to be excluded during statistics.

[0034] Number of inoculated culture dishes: The total number of independent sterile culture containers actually used to carry inoculated anthers in the experimental system.

[0035] Number of contaminated culture dishes: The number of contaminated containers in which visible microbial contamination (such as bacterial colonies, fungal hyphae, etc.) appears during the culture period, resulting in the invalidation of anther data in the culture dish. It is confirmed at the time point when contamination is first detected.

[0036] And calculate the contamination rate, seedling formation rate, browning rate, and induction rate. The calculation methods are as follows: Induction rate = (Number of induced embryos / Number of inoculated anthers (non-contaminated)) × 100% Seedling formation rate = (Number of seedlings / Number of inoculated anthers (non-contaminated)) × 100% Browning rate = (Number of browned anthers / Number of inoculated anthers (non-contaminated)) × 100% Contamination rate = (Number of contaminated culture dishes / Number of inoculated culture dishes) × 100% It should be noted that since the contamination generated during the tissue culture process of pepper anthers is generally exogenous contamination, once contamination occurs, it is very likely that the anthers in the entire culture dish will be contaminated. And because it is easy to cause secondary contamination during the transfer when separately determining whether the anthers are contaminated, the present invention uses the method of calculating the number of contaminated culture dishes to calculate the contamination rate, and the contamination rate measured by this method is representative.

[0037] The statistical results are as Figure 1As shown, the results indicate that ppm, penicillin, and BABA have a negative impact on embryo induction, with the induction rate being lower than that of the control group. In contrast, GABA shows a significant reduction in the contamination rate, along with a significant decrease in the browning rate. At 25 mM, the induction rate is 1.711%, lower than that of the control (2.415%). However, at 50 mM, the embryoid induction rate is significantly increased by 38% compared to the control, the browning rate is decreased by 95.90% compared to the control, and the contamination rate is decreased by 77.46%.

[0038] Example 2 The pepper variety used: B23-5, a linear pepper, scientific name Capsicum annuum L, with the seed source from Jiangsu. The plant height is 89 - 92 cm, the plant width is 87 - 89 cm. The fruits are green, long, and linear. The fruit setting is concentrated, and it is medium-late maturing. The green fruits are green, the red fruits are bright red. The fruit shape index (the ratio of fruit longitudinal diameter to transverse diameter) is 19.7 - 25.3. The capsaicin content is 2.1 - 2.5 mg / g, and the crude fat content is 1.2 - 1.4 mg / g.

[0039] Verify the anther tissue culture effect of the method of the present invention on peppers of other genotypes, and conduct a blank control group during the same period. The specific method is as follows: 1) Pepper cultivation management and flower bud treatment: Carry out plug seedling of the B23-5 pepper variety in the plant culture room in January 2023; the seedlings are planted in the plastic greenhouse using a soilless cultivation mode of foam planting trough + non-woven fabric + nutrient substrate. Lay a 288-hole water-permeable tray at the bottom of the foam planting trough to control the water; lay a layer of non-woven fabric with good water permeability and durability on it; fill the substrate above the non-woven fabric. Before filling, mix perlite and ternary compound fertilizer into the substrate according to a volume ratio of 1:3, and the depth of the substrate soil layer is 35 - 40 cm. The water and fertilizer management uses drip irrigation combined with a proportional pump, and the drip irrigation EC value of the water-soluble fertilizer (product of Chengdu Institute) is controlled at 1.5 - 2.5 mS / cm. When planting, the seedlings need to be planted near the drip irrigation holes to ensure sufficient water absorption. At the initial stage of flower appearance at the end of March, add a certain proportion of GABA (CAS No.: 56-12-2 A800350-100g) of 0.2 - 0.5% to the water-soluble fertilizer. Start picking flower buds at the end of April, and the picking times are from 7 to 8 am from April to May and from 6 to 7 am from May to June. The flower buds are required to have a petal to sepal ratio of about 1, a flower bud length of 2 - 3 mm, and 1 / 3 of the anthers being purple. Pick the flower buds according to the material numbers, pack them well in a plastic-sealed bag, and bring them back to the laboratory in an ice box.

[0040] 2) Bud sterilization: The operations of sterilization and inoculation of flower buds are carried out on the super clean bench in the tissue culture room (Note: The ultraviolet lamp of the super clean bench has been turned on for disinfection 30 minutes in advance). The process of bud treatment is carried out on ice. Bud stripping: Prepare a sterilized stainless steel tray, lay sterilized filter paper in the tray, and place ice cubes at the bottom of the tray. Take out the flower buds in the plastic sealed bag, gently remove the outer sepals with tweezers, and put them into a sterilized tissue culture bottle, preparing for sterilization and disinfection. Bud sterilization: The peeled flower buds are disinfected with an appropriate amount of 75% alcohol (submerging the flower buds) for 30 seconds and shaken. Take out the supernatant, add 0.1% mercuric chloride, and add 1 drop of Tween 80 (15 drops for 100 ml solution), shake for 7 minutes, and finally wash with sterilized water multiple times until there are no bubbles, obtaining sterilized pepper flower buds.

[0041] 3) Anther inoculation and heat shock treatment: Divide the sterilized pepper flower buds into two parts. For one part, carefully take out the anthers from the sterilized flower buds with pointed tweezers and inoculate them into the prepared induction medium culture dish. Inoculate 20 - 30 anthers in each dish, and then seal it with parafilm in two layers. Put the inoculated culture dishes into a 35°C constant temperature incubator for a one - week dark heat shock treatment. Then transfer the culture dishes to a dark culture room at 25 - 28°C for 20 - 30 days. During this period, observe and record the occurrence of embryoids every 7 days. Then carry out light culture. Cultivate the culture dishes in the light culture room, using T8 plant growth lamps as the light source, with a light intensity of 3500 - 5000 LUX, long - day (photoperiod: 16 h light, 8 h dark); the humidity is between 50% - 70%. When the embryoids on the inoculated anthers grow out, transfer them to the culture dish of the embryoid medium to continue growing, with 5 - 15 embryoids in each dish. At this time, the light intensity should be ≥5500; when young roots and young leaves develop, transfer them to the culture bottle of the seedling growth medium to continue growing (light culture), with 1 seedling in each dish. The formula of the medium is as follows: Induction medium: Embryoid (development) medium: Seedling growth medium: The other part of the pepper flower buds is used as a blank control for the same operation. The difference is that the GABA plant resistance inducer is not added to the used induction medium, embryoid medium, and seedling growth medium.

[0042] 4) Hardening off of tissue - cultured seedlings and soil - cultivation management: Backup and acclimatization of tissue-cultured seedlings: To ensure the survival rate of seedlings and subsequent identification work, it is necessary to back up the seedlings. When the seedlings grow to 5 - 8 cm in length, open the bottle cap on the ultra-clean workbench, cut off the apical bud 1.5 - 2.5 cm from the top of the seedling, and remove the leaves, leaving only the apical meristem and the stem. Inoculate the treated apical bud into a new seedling growth medium for light culture, and change the medium monthly. When both the original tissue-cultured seedlings and the backup seedlings have grown new leaves and new roots and become seedlings, they can be acclimatized.

[0043] During acclimatization, to adapt to the microenvironment of the culture room, first loosen the bottle cap of the original tissue-cultured seedlings to allow environmental air to enter the bottle, and harden off for 7 - 10 days. Then, open the bottle cap and add an appropriate amount of pure water to the bottle, adapt for 2 - 7 days until the seedlings are healthy and well-adapted, and then transplant.

[0044] Soil cultivation transplantation and management: First, remove the medium carried on the tissue-cultured seedlings after acclimatization. Gently rinse the roots of the tissue-cultured seedlings, avoiding damaging the roots, and rinse them clean with warm water at 25 - 35°C. Then, soak the rinsed tissue-cultured seedlings for 10 - 20 minutes in a mixed solution containing carbendazim (800 - 1000 times) and rooting powder (20 - 40 mg / kg), and then carry out soil cultivation transplantation. After transplantation, cover with a film or plastic bag to maintain the humidity at 80 - 95%. Add perlite (1:3) to the soil cultivation substrate, supplement the substrate with MS nutrient solution, and remove the covering film after hardening off for 10 - 15 days. Subsequently, enter the daily management.

[0045] Experimental Example 2 Count the number of induced embryos, number of inoculated anthers, number of seedlings, number of browned anthers, number of inoculated culture dishes, and number of contaminated culture dishes during the cultivation process of Example 2 and its blank control experimental group, and calculate the contamination rate, browning rate, and induction rate. The calculation methods are as follows: The statistical results are as follows: The above are only the preferred embodiments of the present invention. It should be understood that the present invention is not limited to the form disclosed herein, should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications, and environments, and can be changed within the scope of the concept described herein through the above teachings or the technology or knowledge in related fields. And the changes and alterations made by those skilled in the art without departing from the spirit and scope of the present invention should all be within the protection scope of the appended claims of the present invention.

Claims

1. A method for reducing the contamination rate of pepper anther tissue culture, characterized in that: It is cultured using a culture medium containing GABA.

2. The method according to claim 1, characterized in that The culture medium includes: an induction medium for inducing the formation of embryoids, an embryoid medium for cultivating embryoids into seedlings, and a seedling growth medium for cultivating seedlings to maturity.

3. The method according to claim 2, characterized in that: The addition amount of GABA in the induction medium, embryoid medium and seedling growth medium is 50 mM.

4. The method according to claim 1, wherein It includes the following steps: S1: Inoculate pepper anthers on the induction medium for culture until embryoids grow on the pepper anthers to obtain embryoids; the induction medium includes MS medium, sucrose, activated carbon, agar powder, silver nitrate, 2,4-D, 6-BA and GABA; S2: Transfer the embryoids to the embryoid medium for culture until the embryoids develop young roots and young leaves to obtain seedling embryoids; the embryoid medium includes MS medium, sucrose, agar powder, silver nitrate, 2,4-D, 6-BA and GABA; S3: Transfer the seedling embryoids to the seedling growth medium for culture until a regenerated plant is obtained; the seedling growth medium includes MS medium, sucrose, agar powder, IBA and GABA.

5. The method according to claim 4, characterized in that: In step S1, the method for culturing the pepper anthers is specifically: perform dark heat shock treatment at 33-36 °C for 6-8 days, then perform dark culture at 25-28 °C for 20-30 days, and finally perform light culture until embryoids grow on the anthers.

6. The method according to claim 4, wherein: By weight, the induction medium includes 4.43 parts of MS medium, 30 parts of sucrose, 0.4 part of activated carbon, 7 parts of agar powder and 10.3 parts of GABA; The induction medium also includes: 4 mg / L silver nitrate, 2,4-D and 0.5 mg / L of 6-BA.

7. The method according to claim 4, characterized in that: By weight, the embryoid medium includes 4.43 parts of MS medium, 30 parts of sucrose, 7 parts of agar powder and 5.15 parts of GABA 0.5 mg / L; The embryoid medium also includes: 4 mg / L silver nitrate, 0.5 mg / L of 2,4-D and 0.5 mg / L of 6-BA.

8. The method according to claim 4, characterized in that: By weight, the seedling growth medium includes 4.43 parts of MS medium, 30 parts of sucrose, 7 parts of agar powder and 5.15 parts of GABA; The seedling growth medium also includes 0.1 mg / L of IBA.

9. The method according to claim 4, wherein The method for obtaining the pepper anthers is: At the initial stage of pepper flowering, add a fertilizer containing GABA for cultivation, then pick the flower buds, disinfect and sterilize them with alcohol, and then take out the pepper anthers from the flower buds.

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