A method for reducing vitrification of anther culture regenerated plants
By covering the mouth of the anther culture bottle with two layers of sealing film, the problem of high vitrification rate in eggplant anther culture was solved, thereby reducing the regeneration rate of vitrified plants and simplifying the production process.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- BEIJING HAIDIAN DISTRICT PLANT TISSUE CULTURE TECH LAB
- Filing Date
- 2024-10-17
- Publication Date
- 2026-05-12
AI Technical Summary
During the development of embryoids in eggplant anther culture, the proportion of malformed and vitrified seedlings is high, resulting in a low proportion of normal plants and affecting the large-scale production of haploid plants.
Cover the mouth of the anther culture bottle with two layers of sealing film. First, tie it tightly with a breathable sealing film, and then cover it with an airtight sealing film. After high temperature treatment, remove the airtight sealing film to reduce humidity changes and avoid the formation of vitrified seedlings.
It reduced the regeneration rate of vitrified plants, simplified the large-scale production process of haploid plants, and reduced the workload.
Smart Images
Figure BDA0005088581250000041
Abstract
Description
Technical Field
[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to a method for reducing vitrification in anther culture-regenerated plants. Background Technology
[0002] Haploid breeding can significantly accelerate the crop breeding process and plays an important role in hybridization breeding. Anther culture is an important method for obtaining haploid plants. In eggplant, haploid induction is highly genotype-dependent, with a low embryoid induction rate. During embryoid development, the proportion of malformed and vitrified seedlings is relatively high, resulting in a low proportion of normal plants, which seriously hinders the widespread application of this technology.
[0003] In obtaining haploid plants, anther culture requires stress treatment in the early stages. Only under certain stress conditions can the metabolism of certain substances in immature pollen be altered, promoting dedifferentiation and deviating from the normal gametophyte development pathway to sporophyte development, forming embryoids, and ultimately developing into complete plants. The typical stress treatment method involves placing the culture bottles containing the anthers at 35℃~38℃ for 3~8 days. During this time, the culture bottles need to be sealed with an airtight film to ensure that the air inside maintains a certain humidity under high temperature conditions, thus not affecting the survival of immature pollen in the anther chambers. If a breathable sealing film is used, moisture inside the bottle will easily evaporate under high temperature conditions, becoming too dry and potentially causing immature pollen to die from dehydration or hindering its dedifferentiation and redifferentiation. After the stress treatment is completed, the plants are transferred to a light source at 25℃~28℃ for another 20~25 days. When embryoids are visibly formed, the anthers and induced embryoids need to be transferred to culture bottles covered with a breathable sealing film. This allows the culture bottles to breathe, reduces humidity, and prevents vitrified seedlings. To further reduce vitrified seedlings, all anthers and induced embryoids must be transferred from airtight culture bottles to breathable ones after 20~25 days of anther culture, significantly increasing the workload. Large-scale production of haploid plants typically requires simplifying the operational process as much as possible without affecting the plant regeneration rate. Summary of the Invention
[0004] The first objective of this invention is to disclose a method for reducing vitrification in anther culture-regenerated plants.
[0005] The objective of this invention is achieved through the following technical solution:
[0006] A method for reducing vitrification in anther culture-regenerated plants includes the following steps:
[0007] (1) Inoculate the anthers into a culture bottle containing culture medium, cover the mouth of the culture bottle with a breathable sealing film, and tie it tightly with a rubber band;
[0008] (2) Then cover the outer layer of the breathable sealing film with another layer of airtight sealing film and tie it tightly with a rubber band;
[0009] (3) After culturing for 20 to 25 days, when embryoids are induced, remove the airtight sealing film covering the embryo, leaving only the airtight sealing film.
[0010] The method described in the above technical solution further includes the step of obtaining anthers.
[0011] The method described in the above technical solution, wherein the step of obtaining the anthers includes:
[0012] (a) Selection of fresh flower buds: Flower buds with immature pollen in the uninucleate marginal stage were selected, and the pollen development stage was determined to be the uninucleate marginal stage by DAPI staining;
[0013] (b) Surface disinfection of flower buds and acquisition of sterile anthers: flower buds are soaked in 70% alcohol for 0.5 to 1 minute, soaked in 5% sodium hypochlorite solution for 3 to 5 minutes, rinsed with sterile water 3 to 5 times, and dried with sterile filter paper.
[0014] The present invention has the following beneficial effects:
[0015] 1. Cover the culture bottle with two layers of sealing film. After inoculating the anthers into the culture medium, first cover the mouth of the culture bottle with a layer of breathable sealing film and secure it with a rubber band; then cover this breathable sealing film with a layer of impermeable sealing film. In this way, when the anthers are subjected to high-temperature treatment, the culture medium will not lose moisture due to excessive temperature, and immature pollen will not die from excessive dryness inside the bottle. After 20-25 days of culture, remove the impermeable sealing film, leaving only the breathable sealing film. This ensures that the induced embryoids will not become vitrified due to excessive humidity inside the culture bottle.
[0016] 2. In this invention, the transformation of the culture bottle from airtight to airtight can be achieved simply by removing the airtight sealing film covering it. It is not necessary to transfer all anthers and the resulting embryoids from the airtight culture bottle to the airtight culture bottle, which greatly reduces the workload of large-scale production of haploid plants. Detailed implementation method:
[0017] To facilitate understanding of the technical solution of the present invention, the following detailed description of a method for reducing vitrification of anther culture-regenerated plants is provided in conjunction with specific embodiments.
[0018] Example 1: A method to reduce vitrification in anther culture-regenerated plants:
[0019] 1. A method for reducing vitrification in anther culture-regenerated plants, comprising the following steps:
[0020] (1) Inoculate the anthers into a culture bottle containing culture medium, cover the mouth of the culture bottle with a breathable sealing film, and tie it tightly with a rubber band;
[0021] (2) Then cover the outer layer of the breathable sealing film with another layer of airtight sealing film and tie it tightly with a rubber band;
[0022] (3) After culturing for 20 to 25 days, when embryoids are induced, remove the airtight sealing film covering the embryo, leaving only the airtight sealing film.
[0023] Both the non-breathable and breathable sealing films were purchased from Yishengshimu (Beijing) Biotechnology Co., Ltd.
[0024] The innovation of this invention lies in covering the culture bottle with two layers of sealing film. After the anthers are inoculated into the culture medium, a breathable sealing film is first placed over the mouth of the culture bottle and secured with a rubber band. Then, an impermeable sealing film is placed on top of this breathable film. During high-temperature treatment of the anthers, the culture medium will not lose moisture due to excessive temperature, and immature pollen will not die from excessive dryness inside the bottle. After 20-25 days of culture, the impermeable sealing film is removed, leaving only the breathable sealing film. This ensures that the induced embryoids will not become vitrified due to excessive humidity inside the culture bottle, significantly reducing the workload for large-scale production of haploid plants.
[0025] The following specific experimental examples illustrate the beneficial effects of the method described in Example 1:
[0026] Experimental Example 1: Eggplant anther culture:
[0027] Experimental steps:
[0028] In June 2022, flower buds from five different types of eggplant were retrieved from the greenhouse.
[0029] 1. Selection of fresh flower buds: Select flower buds with immature pollen in the uninucleate marginal stage, and determine the pollen development stage as uninucleate marginal stage by DAPI staining.
[0030] 2. Surface disinfection of flower buds and obtaining sterile anthers: Soak flower buds in 70% alcohol for 0.5 to 1 minute, soak in 5% sodium hypochlorite solution for 3 to 5 minutes, rinse with sterile water 3 to 5 times, and dry with sterile filter paper; carefully peel the anthers from the flower buds with tweezers.
[0031] 3. Obtaining embryoids: Anthers were inoculated into 50ml Erlenmeyer flasks containing 25ml-30ml of modified 1 / 2 MS medium. The modified 1 / 2 MS medium consisted of 950mg / L KNO3, 825mg / L NH4NO3, 166mg / L CaCl2, 90mg / L MgSO4, 85mg / L KH2PO4, 36.7mg / L FeNaEDTA, 0.83mg / L KI, 6.2mg / L H3BO3, 16.9mg / L MnSO4·H2O, 8.6mg / L ZnSO4·7H2O, 0.25mg / L Na2MoO4·2H2O, 0.025mg / L CuSO4·5H2O, and 0.025mg / L Fe2MoO4·5H2O. The solid culture medium contains CoCl2·6H2O, 100 mg / L inositol, 5 mg / L nicotinic acid, 2 mg / L glycine, 0.5 mg / L thiamine hydrochloride (B1), 0.5 mg / L pyridoxine hydrochloride (B6), 0.5 mg / L folic acid, 0.05 mg / L biotin, 800 mg / L glutamine, 100 mg / L serine, 30 mg / L glutathione (GSH), 30 g / L sucrose, 1–2 mg / L KT, and 0.1%–0.3% activated carbon, with a pH of 5.8.
[0032] Each bottle contains approximately 20 anthers, divided into a pre-optimization group and a post-optimization group, wherein:
[0033] The optimization method for the first group was as follows: anthers were inoculated into Erlenmeyer flasks, and the mouth of the flasks was covered with an airtight sealing film and secured with a rubber band. After incubation in the dark at 33℃~37℃ for 2~8 days, followed by light at 25℃~28℃ for 20~25 days, the anthers and embryoids cultured in the original Erlenmeyer flasks were transferred to Erlenmeyer flasks containing the same culture medium and covered with an airtight sealing film for continued culture, eventually yielding regenerated plants.
[0034] The optimized method was as follows: anthers were inoculated into Erlenmeyer flasks. A breathable sealing film was first placed over the flask opening and secured with a rubber band. Then, an impermeable sealing film was placed over the breathable film and secured with another rubber band. After incubation in the dark at 33℃–37℃ for 2–8 days, the flasks were transferred to a light environment at 25℃–28℃ for 20–25 days. When the embryoids germinated from the split anther walls, the outer impermeable sealing film was removed, and the culture continued until regenerated plants were obtained.
[0035] 4. The improved anther culture method can significantly reduce the regeneration rate of vitrified plants. Table 1 shows the comparison of embryoid induction rate and vitrified plant regeneration rate before and after optimization of eggplant anther culture.
[0036] Table 1 Comparison of embryoid induction rate and vitrified plant regeneration rate before and after optimization of eggplant anther culture.
[0037]
[0038] Note: Different lowercase letters in the same row indicate significant differences at the P<0.05 level.
[0039] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention in any form or substance. Any modifications, alterations, and variations made by those skilled in the art without departing from the scope of the present invention using the disclosed technical content are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and variations made to the above embodiments based on the essential technology of the present invention are still within the scope of the present invention.
Claims
1. A method for reducing vitrification in eggplant anther culture regenerated plants, comprising the following steps: (1) Inoculate the eggplant anthers into a culture bottle containing culture medium, cover the mouth of the culture bottle with a breathable sealing film, and tie it tightly with a rubber band; (2) Then cover the outer layer of the breathable sealing film with another layer of airtight sealing film and tie it tightly with a rubber band; (3) After culturing for 20 to 25 days, when embryoids are induced, remove the airtight sealing film covering the embryo, leaving only the airtight sealing film.
2. The method according to claim 1, characterized in that, The method also includes the step of obtaining anthers.
3. The method according to claim 2, characterized in that, The steps for obtaining anthers include: (a) Selection of fresh flower buds: Flower buds with immature pollen in the uninucleate marginal stage were selected, and the pollen development stage was determined to be the uninucleate marginal stage by DAPI staining; (b) Surface disinfection of flower buds and obtaining sterile anthers: flower buds are soaked in 70% alcohol for 0.5 to 1 minute, soaked in 5% sodium hypochlorite solution for 3 to 5 minutes, rinsed with sterile water 3 to 5 times, and dried with sterile filter paper.