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

Abstract

The present invention relates to the technical field of pepper anther tissue culture, and specifically discloses a method for increasing the embryoid induction rate and reducing the contamination rate in pepper anther tissue culture. The method comprises: using a culture medium containing GABA for culturing. The culture medium specifically comprises: an induction medium for inducing embryoid formation, an embryoid culture medium for cultivating embryoids into seedlings, and a seedling growth medium for cultivating mature seedlings. Based on conventional culture medium formulas, the present invention adds 50 mM GABA to the culture medium at each stage, significantly increasing the embryoid induction rate and reducing the contamination rate, ultimately achieving the purpose of increasing the success rate of pepper anther culture. Specifically, during the anther tissue culture process, the culture medium supplemented with 50 mM GABA reduced the contamination rate by 4.5 to 2.5 times compared to conventional culture medium, increased the embryoid induction rate by an average of 1.6 to 2.2 times, and increased the number of surviving haploid seedlings by 1.6 to 3.3 times.

Description

Technical Field

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

[0002] Pepper (Capsicum annum L.) is an important Solanaceae vegetable crop in my country, widely cultivated throughout the country. Pepper breeding research in my country is of great significance. First, as a key vegetable crop, pepper enjoys enormous domestic demand, particularly in Southwest China and Central China. Pepper is an indispensable condiment in daily diets, and the market demands diverse varieties, tastes, and spiciness, driving the diversification of breeding research.

[0003] Pepper breeding methods include traditional breeding (selection breeding, hybrid breeding, and backcross breeding), modern biotechnology breeding (marker-assisted selection, genetic engineering, and cell engineering), mutation breeding, polyploid breeding, genomics-assisted breeding, and phenomics-assisted breeding. These methods, when combined, can efficiently produce disease-resistant, stress-tolerant, high-yielding, and high-quality pepper varieties. Among them, haploid breeding using anther culture plays a crucial role in pepper breeding. By inducing microspores or pollen to form haploid plants and doubling chromosomes, haploid breeding can rapidly produce homozygous lines, significantly shortening the breeding cycle. This technique improves selection efficiency, facilitates the fixation of target traits, and screens for recessive, superior genes. It also promotes genetic improvement and germplasm innovation, enriching genetic diversity. Due to its significant advantages in disease resistance breeding and quality improvement, haploid breeding using anther culture can efficiently and precisely produce disease-resistant, high-quality, and high-yielding pepper varieties. Therefore, anther culture is an indispensable key technology in pepper breeding.

[0004] Anther culture holds great potential for rapid propagation and genetic improvement of pepper. By inducing haploid plants, anther culture can produce fully homozygous inbred lines within a single generation, significantly shortening the number of inbreeding generations required in traditional breeding. This method not only reduces production costs but also significantly improves reproductive efficiency. Furthermore, as a precision breeding tool, anther culture facilitates the development of new pepper varieties with enhanced resistance or adaptability while maintaining genetic stability. These advantages are of great significance for efficient breeding and genetic improvement of pepper. The most critical step in anther culture is embryoid formation, which directly determines the successful induction of haploid plants. To overcome this critical step, several approaches can be taken: In terms of material selection, anthers from the late uninucleate to early dinucleate stage should be selected, with preference given to easily inducible genotypes or genetic modification to increase induction rates. Regarding the culture system, the ratios of hormones, carbon sources, and additives in the culture medium must be carefully optimized. Environmental control requires precise control of temperature, light, and humidity. Furthermore, during the pretreatment phase, low temperatures or chemical treatments can be used to activate the dedifferentiation capacity of the anthers. In addition, when the embryoid germinates, it must be transferred to an adaptive culture medium in a timely manner to maintain a stable environment to ensure smooth plant regeneration. In anther culture, there are two ways to induce embryoid formation. One is to first induce pollen to form callus tissue. Callus tissue is an undifferentiated cell mass, and then the callus tissue differentiates into embryoids. The other is to directly induce embryoids. Direct induction into embryoids has many advantages. It can reduce the number of cell divisions, reduce the risk of mutation, and better maintain genetic stability. There is no need to go through the callus tissue stage, which greatly shortens the culture cycle and accelerates the seedling formation speed. Moreover, the embryoids formed by direct induction have better physiological activity, the developed plants have strong growth potential, the probability of deformed seedlings is low, and the output rate of effective seedlings can be increased.

[0005] Since the first successful induction of haploid embryos from pepper anthers, anther culture has made significant progress in pepper breeding. However, its practical application still faces numerous challenges, including dependence on the donor plant's genotype, the developmental stage of microspores, pretreatment methods, and culture conditions. Genotype dependence and high contamination rates during culture remain major obstacles to successful pepper anther culture.

[0006] Contamination is a common problem in plant tissue culture. During rapid propagation, contamination usually comes from incomplete sterilization of culture media, inoculation tools, clean benches or inoculation rooms, as well as non-standard aseptic techniques during operations. In addition, microorganisms may attach to the surface of plant materials exposed to the environment for a long time, leading to exogenous contamination. Some microorganisms can even invade the intercellular spaces or internal cells of plant tissues. These microorganisms cannot be completely eliminated by surface sterilization, thereby causing endogenous contamination. Once introduced into the culture system, it will seriously affect the culture effect. These pollutants not only reduce the success rate of tissue culture, but also increase production costs. To deal with exogenous contamination, disinfectants such as ethanol and sodium hypochlorite are usually used. These methods can effectively kill pathogens, but the concentration of disinfectants must be strictly controlled to avoid toxicity or damage to plant tissues.

[0007] Existing anther culture technology has the following problems: low embryoid induction rate, higher callus induction rate than embryoid induction rate, and high contamination rate, which affects the formation of embryoid 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 pepper anther tissue culture, so as to at least increase the embryoid induction rate, reduce the contamination rate and ultimately improve the success rate of pepper anther culture.

[0009] The object of the present invention is achieved through the following technical solutions:

[0010] A method for reducing the contamination rate of pepper anther tissue culture: culture is performed using a culture medium containing GABA (γ-aminobutyric acid).

[0011] In some embodiments, the culture medium includes: an induction medium for inducing embryoid formation; an embryoid medium for cultivating embryoids into seedlings; and a seedling growth medium for cultivating seedlings to maturity.

[0012] In some examples, the amount of GABA added to the induction medium, embryoid medium, and seedling growth medium is 50 mM.

[0013] In some other embodiments, the method comprises the following steps:

[0014] S1: inoculating pepper anthers into an induction medium for culturing until embryoids grow out of the pepper anthers to obtain embryoids; the induction medium comprises MS medium, sucrose, activated carbon, agar powder, silver nitrate, 2,4-D, 6-BA and GABA;

[0015] S2: transferring the embryoids to an embryoid culture medium for culturing until the embryoids develop young roots and leaves, thereby obtaining seedling embryoids; the embryoid culture medium comprises MS medium, sucrose, agar powder, silver nitrate, 2,4-D, 6-BA, and GABA;

[0016] S3: transferring the seedling embryoids to a seedling growth medium for culture to obtain regenerated plants; the seedling growth medium comprises MS medium, sucrose, agar powder, IBA and GABA.

[0017] In some examples, the method for culturing pepper anthers in step S1 is specifically: performing dark heat shock treatment at 33-36°C for 6-8 days, then dark culturing at 25-28°C for 20-30 days, and finally light culturing until the anthers grow embryoids.

[0018] In some examples, the induction medium includes, by weight, 4.43 parts of MS medium, 30 parts of sucrose, 2 parts of activated carbon, 7 parts of agar powder, and 5.15 parts of GABA;

[0019] The induction medium also includes: 4 mg / L silver nitrate, 0.5 mg / L 2,4-D and 0.5 mg / L 6-BA.

[0020] In some examples, the embryoid culture medium comprises, by weight, 4.43 parts of MS medium, 30 parts of sucrose, 7 parts of agar powder, and 5.15 parts of GABA;

[0021] The embryoid culture medium also includes: 4 mg / L silver nitrate, 0.5 mg / L 2,4-D and 0.5 mg / L 6-BA.

[0022] In some examples, the seedling growth medium includes, by weight, 4.43 parts of MS medium, 30 parts of sucrose, 7 parts of agar powder, and 5.15 parts of GABA;

[0023] The seedling growth medium also included 0.1 mg / L IBA.

[0024] In some examples, the method for obtaining pepper anthers is:

[0025] At the early stage of pepper flowering, fertilizer containing GABA is added for cultivation, and then the flower buds are picked, disinfected and sterilized with alcohol, and then the pepper anthers are taken out from the flower buds.

[0026] The beneficial effects of the present invention are:

[0027] The present invention, based on a conventional culture medium formula, adds 50 mM GABA to the culture medium at each stage, significantly improving the embryoid induction rate while reducing the contamination rate, ultimately achieving the goal of increasing the success rate of pepper anther culture. During anther tissue culture, the addition of 50 mM GABA to the culture medium reduced the contamination rate by 4.5 to 2.5 times compared to conventional culture medium, increased the embryoid induction rate by an average of 1.6 to 2.2 times, and increased the number of surviving haploid seedlings by 1.6 to 3.3 times. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The calculation results of the contamination rate, seedling rate, browning rate and induction rate of Example 1 and Comparative Examples 1-4 in Experimental Example 1;

[0029] Figure 2 The following are photographs of various stages of pepper tissue culture in the middle section of Example 1; A in the figure is an anther removed from the flower bud at the beginning of culture in step 3); B shows the anther swelling during the induction culture process; C shows the embryoid emerging from the anther during the induction culture process; DF shows different stages of embryoid development, D is an elongated embryoid, E is an embryoid turning green, and F is a cotyledon-type embryo; G is an embryoid transplanted onto the embryoid culture medium; H is an embryoid with gradually developing roots, forming a regenerated plant with roots, stems, and leaves; I is a tissue culture plantlet with 6 to 8 true leaves; J is a plant developed after 2 weeks of growth in soil; K is an anther with abnormal development, gradually turning brown; L is an embryo that has formed callus tissue and cannot continue to develop normally. DETAILED DESCRIPTION

[0030] The technical solution 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.

[0031] The sources of the antibacterial agents used in the following examples and comparative examples are:

[0032] Plant Preservative Mixture (PPMTM, a broad-spectrum antifungal agent), brand: Cimejie; 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: C16H18N2O4S A800090-5g), purchased from Beijing Coupling.

[0033] The pepper variety used is B23-3, Capsicum annuum L., sourced from Surun, Jiangsu Province. Plants are 88-90 cm tall and 86-88 cm wide. The fruit is green, long, linear, with concentrated fruit set and early maturity. Green fruit is green, while red fruit is bright red. The fruit shape index (ratio of longitudinal diameter to transverse diameter) ranges from 18.3 to 24.4, with capsaicin content of 1.8-2.2 mg / g and crude fat content of 0.85-0.92 mg / g.

[0034] Example 1

[0035] Pepper anther tissue culture was performed, and GABA was added as a plant inducer to the induction medium, embryoid medium, and seedling growth medium. The specific method is as follows:

[0036] 1) Pepper Cultivation Management and Bud Treatment: In January 2023, seedlings of the B23-3 pepper variety were raised in a plant cultivation room using plug trays. The seedlings were then transplanted in a plastic greenhouse using a soilless cultivation system consisting of a foam planting trough, non-woven fabric, and a nutrient matrix. A 288-hole plug tray with water holes was placed at the bottom of the foam planting trough to control moisture. A layer of durable, permeable non-woven fabric was placed on top. A matrix was added above the non-woven fabric. Perlite and a three-element compound fertilizer were mixed into the matrix at a volume ratio of 1:3. The matrix depth was 35-40 cm. Drip irrigation with a proportional pump was used for fertilization. The EC value of the water-soluble fertilizer (from the Chengdu Urban Research Institute) was controlled at 1.5-2.5 mS / cm. When transplanting, the seedlings should be planted near the drip irrigation holes to ensure adequate water absorption. At the beginning of flowering in late March, add 0.2-0.5% GABA (CAS number: 56-12-2 A800350-100g) to the water-soluble fertilizer. Flower buds should be harvested starting in late April, between 7:00 and 8:00 AM from April to May and between 6:00 and 7:00 AM from May to June. The ideal petal-to-sepal ratio is approximately 1:1, the buds should be 2-3 mm long, and one-third of the anthers should be purple. Buds should be picked according to their material number, sealed in plastic bags, and placed in an ice box for transport back to the laboratory. 2) Flower Bud Sterilization: Sterilize and inoculate the flower buds on the clean bench in the tissue culture room (note: the clean bench has been pre-treated with UV light for 30 minutes). All bud handling should be performed on ice. Bud Stripping: Prepare a sterilized stainless steel tray, line it with sterilized filter paper, and place ice cubes on the bottom. Remove the flower buds from the plastic bag and gently peel off the outer calyx and sepals with tweezers. Place them in a sterilized tissue culture flask and prepare for sterilization. Flower Bud Sterilization: Sterilize the peeled buds with an appropriate amount of 75% alcohol (covering the buds) for 30 seconds while shaking. Remove the supernatant, add 0.1% mercuric chloride and 1 drop of Tween 80 (15 drops per 100 ml solution), shake for 7 minutes, and finally rinse with sterile water several times until there are no bubbles. This will sterilize the pepper buds.

[0037] 3) Anther Inoculation and Heat Shock Treatment: Carefully remove anthers from sterilized buds using pointed forceps and inoculate 20-30 anthers per dish into a prepared induction medium. Seal the dish twice with parafilm. Place the inoculated dish in a 35°C incubator in the dark for one week for heat shock treatment. Then, transfer the dish to a dark room at 25-28°C for 20-30 days. Observe and record the development of embryoids every 7 days. Then, perform light culture in a light-treated room using T8 plant growth lamps with a light intensity of 3500-5000 LUX and a long-day photoperiod (16 hours of light and 8 hours of dark). Maintain a humidity level between 50% and 70%. When embryoids grow from the inoculated anthers, they are transferred to culture dishes containing embryoid culture medium for further growth, with 5 to 15 embryoids per dish. At this time, the light intensity should be ≥5500. After young roots and leaves develop, they are transferred to culture bottles containing seedling growth medium for further growth (light culture), with one seedling per dish. The medium formula is as follows:

[0038] Induction medium:

[0039]

[0040] Embryoid (Development) Medium:

[0041]

[0042] Seedling growth medium:

[0043]

[0044] 4) Hardening and soil culture management of tissue culture seedlings:

[0045] Backup and hardening of tissue culture seedlings: To ensure seedling survival and subsequent identification, backup seedlings are necessary. When the seedlings are 5-8 cm long, remove the bottle cap on a clean bench and cut the terminal bud 1.5-2.5 cm below the top of the seedling. Remove the leaves, retaining only the tip growth point and stem. Inoculate the treated terminal bud into a new seedling growth medium for light culture, changing the medium monthly. Once both the original tissue culture seedlings and the backup seedlings have grown new leaves and roots, they can be hardened.

[0046] During hardening, to help the seedlings adapt to the microenvironment in the culture room, first loosen the original tissue culture bottle cap to allow ambient air to enter the bottle and allow the seedlings to harden for 7 to 10 days. Then, open the bottle cap and add an appropriate amount of purified water to the bottle. Allow the seedlings to adapt for 2 to 7 days until they are healthy and well adapted before transplanting.

[0047] 5) Soil Culture, Transplanting, and Management: First, remove the culture medium from the tissue culture seedlings after hardening. Gently rinse the roots of the seedlings, avoiding root damage, with warm water at 25-35°C. Then, soak the cleaned seedlings for 10-20 minutes in a solution containing a mixture of carbendazim (800-1000 times diluted) and rooting powder (20-40 mg / kg). Then, transplant them into the soil. After transplanting, cover with film or plastic bags to maintain a humidity of 80-95%. Add perlite (1:3) to the soil culture medium and rehydrate with MS nutrient solution. After 10-15 days of hardening, remove the film and proceed with daily care.

[0048] Comparative Example 1

[0049] Pepper anther tissue culture was carried out using the same method as in Example 1, and was performed at the same time as in Example 1, except that two sets of experiments were conducted simultaneously, with 0.0625% and 0.075% ppm added as a plant inducer to the induction medium, embryoid medium, and seedling growth medium, respectively (the addition amount here is the conventional addition amount recommended by the reagent manufacturer).

[0050] Comparative Example 2

[0051] Pepper anther tissue culture was carried out using the same method as in Example 1, and was performed at the same time as in Example 1, except that two groups of experiments were conducted simultaneously, with BABA added as a plant inducer at 25 mM and 50 mM, respectively, to the induction medium, embryoid medium, and seedling growth medium.

[0052] Comparative Example 3

[0053] Pepper anther tissue culture was performed using the same method as in Example 1, performed concurrently with Example 1, with the difference that two sets of experiments were performed simultaneously, with penicillin added as a plant elicitor at concentrations of 2.50% and 5.00% (of the total culture medium) to the induction medium, embryoid culture medium, and seedling growth medium, respectively (the amount added here is the conventional amount recommended by the reagent manufacturer).

[0054] Experimental Example 1

[0055] The number of induced embryos, inoculated anthers, seedlings, browned anthers, inoculated culture dishes, and contaminated culture dishes during the cultivation process of Example 1 and Comparative Examples 1-3 were counted:

[0056] The term "number of induced embryos" refers to the number of anthers that successfully induced to form typical embryoids in vitro under specific culture medium formulations and conditions. Individual anthers were counted as units, and the formation of a single or multiple embryoids from the same anther was counted as one effective induced embryoid.

[0057] Number of inoculated anthers: The total number of viable anthers isolated from donor plants at the initial stage of the experiment, surface sterilized, and inoculated intact into culture medium for in vitro culture. Ineffective inoculations due to structural damage caused by handling are not included.

[0058] Seedling number: The number of regenerated plants that have completed complete plant differentiation through embryoid regeneration and possess standard morphological characteristics (at least root system and three or more true leaves) suitable for transplantation. Abnormal development of seedlings, such as deformed seedlings and vitrified seedlings, must be excluded.

[0059] Browning anthers: The number of anthers that have lost their regenerative potential due to the oxidative damage of phenolic substances, resulting in a noticeable browning of the culture medium and anthers, accompanied by a loss of cell viability. Samples with localized browning due to mechanical damage should be excluded from the statistics.

[0060] Number of inoculated culture dishes: the total number of independent sterile culture containers actually used to hold inoculated anthers in the experimental system.

[0061] Number of contaminated dishes: The number of contaminated containers with invalid anther data due to visible microbial contamination (e.g., bacterial colonies, fungal hyphae, etc.) during the culture cycle. Confirmed at the time of first discovery of contamination.

[0062] The contamination rate, seedling rate, browning rate and induction rate are calculated as follows:

[0063] Induction rate = (number of induced embryos / number of inoculated anthers (non-contaminated)) × 100%

[0064] Seedling rate = (number of seedlings / number of inoculated anthers (non-contaminated)) × 100%

[0065] Browning rate = (number of browning anthers / number of inoculated anthers (uncontaminated)) × 100%

[0066] Contamination rate = (number of contaminated dishes / number of inoculated dishes) × 100%

[0067] It should be noted that since the contamination generated during the pepper anther tissue culture process is generally exogenous contamination, once contamination occurs, there is a high probability that the anthers of the entire culture dish will be contaminated. In addition, since determining whether the anthers are contaminated alone is likely to cause secondary contamination during transfer, 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.

[0068] The statistical results are as follows Figure 1As shown, the results showed that ppm, penicillin, and BABA negatively affected embryo induction, with induction rates lower than those in the control. In contrast, GABA showed a significant reduction in contamination and browning rates. At 25 mM, the induction rate was 1.711%, lower than the control (2.415%). However, at 50 mM, the embryoid induction rate was significantly increased by 38%, the browning rate was reduced by 95.90%, and the contamination rate was reduced by 77.46%.

[0069] Example 2

[0070] The pepper variety used is B23-5, Capsicum annuum L., sourced from Jiangsu Province. Plants are 89-92 cm tall and 87-89 cm wide. Fruit is long, green, and linear, with concentrated fruit set. It matures in the middle to late seasons. Green fruit is green, while red fruit is bright red. The fruit shape index (ratio of longitudinal diameter to transverse diameter) ranges from 19.7 to 25.3, with capsaicin content of 2.1-2.5 mg / g and crude fat content of 1.2-1.4 mg / g.

[0071] The effect of the method of the present invention on anther tissue culture of other genotype peppers was verified, and a blank control group was implemented at the same time. The specific method is as follows:

[0072] 1) Pepper Cultivation Management and Bud Treatment: In January 2023, seedlings of the B23-5 pepper variety were raised in a plant cultivation room using plug trays. The seedlings were then transplanted in a plastic greenhouse using a soilless cultivation system consisting of a foam planting trough, non-woven fabric, and a nutrient matrix. A 288-hole plug tray with water holes was placed at the bottom of the foam planting trough to control moisture. A layer of durable, permeable non-woven fabric was placed on top. A matrix was added above the non-woven fabric. Perlite and a three-element compound fertilizer were mixed into the matrix at a volume ratio of 1:3. The matrix depth was 35-40 cm. Drip irrigation with a proportional pump was used for water and fertilizer management. The EC value of the water-soluble fertilizer (from the Chengdu Urban Research Institute) was controlled at 1.5-2.5 mS / cm. When transplanting, the seedlings should be planted near the drip irrigation holes to ensure adequate water absorption. At the beginning of flowering in late March, add 0.2-0.5% GABA (CAS number: 56-12-2 A800350-100g) to water-soluble fertilizer. Flower buds should be harvested starting in late April, between 7:00 and 8:00 a.m. from April to May and between 6:00 and 7:00 a.m. from May to June. Ideally, the buds should have a petal-to-calyx ratio of approximately 1, be 2-3mm long, and have one-third of the anthers purple. Buds should be harvested according to their material number, sealed in plastic bags, and placed in an ice box for transport back to the laboratory.

[0073] 2) Flower Bud Sterilization: Sterilize and inoculate flower buds on a clean bench in the tissue culture room (Note: The clean bench has been pre-treated with UV light for 30 minutes). All flower bud handling procedures are performed on ice. Flower Bud Stripping: Prepare a sterilized stainless steel tray, line it with sterilized filter paper, and place ice cubes on the bottom. Remove the flower buds from the plastic bag and gently peel off the outer calyx and sepals with tweezers. Place the buds in a sterilized tissue culture bottle and prepare for sterilization. Flower Bud Sterilization: Disinfect the stripped buds with an appropriate amount of 75% alcohol (covering the buds) for 30 seconds and shake. Remove the supernatant, add 0.1% mercuric chloride and 1 drop of Tween 80 (15 drops per 100 ml of solution), and shake for 7 minutes. Finally, rinse with sterile water several times until no bubbles appear. This will yield sterilized pepper flower buds.

[0074] 3) Anther Inoculation and Heat Shock Treatment: Split the sterilized pepper buds into two portions. Carefully remove anthers from the sterilized buds using pointed tweezers and inoculate 20-30 anthers per dish into a culture dish containing the prepared induction medium. Seal the two dishes with parafilm. Place the inoculated dishes in a 35°C incubator in the dark for one week for heat shock treatment. Then, transfer the dishes to a dark room at 25-28°C for 20-30 days. Observe and record the development of embryoids every 7 days. Then, perform light culture in a light-treated room using T8 plant growth lamps with a light intensity of 3500-5000 LUX and a long-day photoperiod (16 hours of light and 8 hours of dark). Humidity should be maintained between 50% and 70%. When embryoids grow from the inoculated anthers, they are transferred to culture dishes containing embryoid culture medium for further growth, with 5 to 15 embryoids per dish. At this time, the light intensity should be ≥5500. After young roots and leaves develop, they are transferred to culture bottles containing seedling growth medium for further growth (light culture), with one seedling per dish. The medium formula is as follows:

[0075] Induction medium:

[0076] Embryoid (Development) Medium:

[0077]

[0078] Seedling growth medium:

[0079]

[0080] Another pepper bud was subjected to the same operation as a blank control, except that no GABA plant elicitor was added to the induction medium, embryoid medium and seedling growth medium used.

[0081] 4) Hardening and soil culture management of tissue culture seedlings:

[0082] Backup and hardening of tissue culture seedlings: To ensure seedling survival and subsequent identification, backup seedlings are necessary. When the seedlings are 5-8 cm long, remove the bottle cap on a clean bench and cut the terminal bud 1.5-2.5 cm below the top of the seedling. Remove the leaves, retaining only the tip growth point and stem. Inoculate the treated terminal bud into a new seedling growth medium for light culture, changing the medium monthly. Once both the original tissue culture seedlings and the backup seedlings have grown new leaves and roots, they can be hardened.

[0083] During hardening, to help the seedlings adapt to the microenvironment in the culture room, first loosen the original tissue culture bottle cap to allow ambient air to enter the bottle and allow the seedlings to harden for 7 to 10 days. Then, open the bottle cap and add an appropriate amount of purified water to the bottle. Allow the seedlings to adapt for 2 to 7 days until they are healthy and well adapted before transplanting.

[0084] Soil culture, transplanting, and care: First, remove the culture medium from the tissue culture seedlings after hardening. Gently rinse the roots, avoiding root damage, with warm water at 25-35°C. Then, soak the cleaned seedlings for 10-20 minutes in a solution containing a mixture of carbendazim (800-1000 times diluted) and rooting powder (20-40 mg / kg). Then, soil culture and transplanting are performed. After transplanting, cover with film or plastic bags to maintain a humidity of 80-95%. Add perlite (1:3) to the soil culture medium and rehydrate with MS nutrient solution. After 10-15 days of hardening, remove the film and proceed with daily care.

[0085] Experimental Example 2

[0086] The number of induced embryos, inoculated anthers, seedlings, browned anthers, inoculated culture dishes, and contaminated culture dishes during the cultivation process of Example 2 and its blank control experimental group were counted, and the contamination rate, browning rate, and induction rate were calculated as follows:

[0087] The statistical results are as follows:

[0088]

[0089] The foregoing description is merely a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the form disclosed herein and should not be construed as excluding other embodiments. Rather, the present invention can be used in various other combinations, modifications, and environments and can be modified within the scope of the concept described herein through the above teachings or techniques or knowledge in the relevant field. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention are intended to be protected by the appended claims.

Claims

1. A method for reducing the contamination rate of pepper anther tissue culture, characterized in that: Cultured in a medium containing GABA; an induction medium for inducing embryoid formation, an embryoid medium for cultivating embryoids into seedlings, and a seedling growth medium for cultivating seedlings to maturity; The amount of GABA added to the induction medium, embryoid medium and seedling growth medium is 50 mM; By weight, the induction medium includes 4.43 parts of MS medium, 30 parts of sucrose, 2 parts of activated carbon, 7 parts of agar powder and 5.15 parts of GABA; The induction medium also includes: 4 mg / L silver nitrate, 0.5 mg / L 2,4-D and 0.5 mg / L 6-BA; The embryoid culture medium comprises, by weight, 4.43 parts of MS medium, 30 parts of sucrose, 7 parts of agar powder, and 5.15 parts of GABA; The embryoid culture medium further comprises: 4 mg / L silver nitrate, 0.5 mg / L 2,4-D and 0.5 mg / L 6-BA; The seedling growth medium comprises, by weight, 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 included 0.1 mg / L IBA.

2. The method according to claim 1, characterized in that The following steps are involved: S1: inoculating pepper anthers into an induction medium for culturing until embryoids grow out of the pepper anthers to obtain embryoids; the induction medium comprises MS medium, sucrose, activated carbon, agar powder, silver nitrate, 2,4-D, 6-BA and GABA; S2: transferring the embryoids to an embryoid culture medium for culturing until the embryoids develop young roots and leaves, thereby obtaining seedling embryoids; the embryoid culture medium comprises MS medium, sucrose, agar powder, silver nitrate, 2,4-D, 6-BA, and GABA; S3: transferring the seedling embryoids to a seedling growth medium for culture until regenerated plants are obtained; the seedling growth medium comprises MS medium, sucrose, agar powder, IBA and GABA.

3. The method according to claim 2, wherein: In step S1, the method for culturing pepper anthers is specifically: performing dark heat shock treatment at 33-36°C for 6-8 days, then dark culturing at 25-28°C for 20-30 days, and finally performing light culturing until the anthers grow embryoids.

4. The method according to claim 2, characterized in that The method for obtaining the pepper anthers is as follows: At the early stage of pepper flowering, fertilizer containing GABA is added for cultivation, and then the flower buds are picked, disinfected and sterilized with alcohol, and then the pepper anthers are taken out from the flower buds.

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