CN-119014328-B - Method for reducing vitrification of anther culture regenerated plants

Abstract

The invention discloses a method for reducing vitrification of regenerated plants in anther culture, belonging to the technical field of plant tissue culture. The method comprises the steps of inoculating anther to a culture bottle containing a culture medium, covering a layer of air-permeable sealing film on the bottle mouth of the culture bottle, fastening by using a rubber band, covering a layer of air-impermeable sealing film on the outer layer of the air-permeable sealing film, fastening by using the rubber band, and removing the covered air-impermeable sealing film and only leaving the air-permeable sealing film when embryoid is induced for 20-25 days. The method has the advantages that when anther is subjected to high-temperature treatment, the culture bottle is covered by the airtight sealing film, the humidity in the bottle is not too low, the survival of immature pollen is not affected, when embryoid is induced for 20-25 days, the airtight sealing film is removed, only the airtight sealing film is remained, vitrified seedlings are not produced by the embryoid due to overlarge humidity, and the proper humidity in the culture bottle can be realized only by covering the airtight sealing film or not, so that the workload of large-scale production of haploid plants is reduced.

Inventors

• DENG XIAOMEI
• ZHANG QIN
• YANG HUIMIAO
• HUI ZHIMING
• ZHANG JUNMIN

Assignees

• 北京市海淀区植物组织培养技术实验室
• 北京海花生物科技有限公司

Dates

Publication Date

20260512

Application Date

20241017

Claims (3)

1. 1. A method for reducing vitrification of regenerated plants in anther culture of eggplants, comprising the following steps: (1) Inoculating eggplant anther into a culture bottle containing a culture medium, covering a layer of breathable sealing film on the bottle mouth of the culture bottle, and fastening by using rubber bands; (2) Then covering a layer of airtight sealing film on the outer layer of the breathable sealing film, and fastening by using a rubber band; (3) Culturing for 20-25 days, and removing the covered airtight sealing film when embryoid is induced, and only leaving the airtight sealing film.
2. 2. The method of claim 1, further comprising the step of obtaining an anther.
3. 3. The method of claim 2, wherein the step of obtaining anthers comprises: (a) Selecting fresh flower buds, namely selecting flower buds of immature pollen in a mononuclear borderline, and determining that the pollen development period is the mononuclear borderline through DAPI dyeing; (b) The method comprises the steps of sterilizing the surfaces of flower buds and obtaining sterile anthers, wherein the flower buds are soaked in 70% alcohol for 0.5-1 min, soaked in 5% sodium hypochlorite solution for 3-5 min, washed for 3-5 times with sterile water, and the sterile filter paper absorbs water.

Description

Method for reducing vitrification of anther culture regenerated plants Technical Field The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for reducing vitrification of regenerated plants in anther culture. Background Haploid breeding can significantly accelerate crop breeding processes, and plays an important role in cross breeding. Anther culture is an important way to obtain haploid plants. The eggplant has serious genotype dependence in the haploid induction process, low embryoid induction rate and larger malformed seedlings and vitrified seedlings in the embryoid development process, so that the normal plant proportion is lower, and the wide application of the technology is seriously hindered. In the process of obtaining haploid plants, anther needs to be subjected to stress treatment in the early stage of anther culture, and the immature pollen can be promoted to start dedifferentiation only under certain stress conditions by changing the metabolism of certain substances, deviates from the normal gametophyte development path and turns to sporophyte development to form embryoids, so that complete plants are developed. The common stress treatment method is to treat the culture bottle with anther at 35-38 ℃ for 3-8 days. At this time, the culture bottle needs to use an airtight sealing film to ensure that the air in the bottle keeps certain humidity under the high-temperature condition, and the survival of immature pollen in the anther chamber is not influenced. If the breathable sealing film is used, water in the bottle is easy to be lost under the high-temperature condition, and the bottle is too dry, so that premature pollen is easy to lose water and die, or the dedifferentiation and the redifferentiation of the pollen are influenced. And after the stress treatment is finished, the culture is continued for 20-25 days under the illumination of 25-28 ℃, and when embryoid generation is seen by naked eyes, the anther and the embryoid generated by induction are required to be transferred into a culture bottle covered with an air-permeable sealing film, so that the culture bottle is air-permeable, the humidity in the bottle is reduced, and vitrified seedlings are not easy to generate. In order to reduce the generation of glass seedlings, when anthers are cultured for 20-25 days, all anthers and embryoids generated by induction are required to be transferred from an airtight culture bottle to an air-permeable culture bottle, so that the workload is greatly increased. The large-scale production of haploid plants generally requires simplifying the operation flow as much as possible under the condition of not affecting the plant regeneration rate. Disclosure of Invention A first object of the present invention is to disclose a method for reducing vitrification of anther-cultured regenerated plants. The invention aims at realizing the following technical scheme: a method for reducing vitrification of anther-cultured regenerated plants comprising the steps of: (1) Inoculating anther into a culture bottle containing a culture medium, covering a layer of breathable sealing film on the bottle mouth of the culture bottle, and fastening by using rubber bands; (2) Then covering a layer of airtight sealing film on the outer layer of the breathable sealing film, and fastening by using a rubber band; (3) Culturing for 20-25 days, and removing the covered airtight sealing film when embryoid is induced, and only leaving the airtight sealing film. The method according to the above technical scheme, wherein the method further comprises the step of acquiring anthers. The method of the above technical scheme, wherein the step of obtaining anther comprises the following steps: (a) Selecting fresh flower buds, namely selecting flower buds of immature pollen in a mononuclear borderline, and determining that the pollen development period is the mononuclear borderline through DAPI dyeing; (b) The method comprises the steps of sterilizing the surfaces of flower buds and obtaining sterile anthers, wherein the flower buds are soaked in 70% alcohol for 0.5-1 min, soaked in 5% sodium hypochlorite solution for 3-5 min, washed for 3-5 times with sterile water, and the sterile filter paper absorbs water. The invention has the following beneficial effects: 1. The culture flask was covered with two sealing films. The anther is inoculated to the culture medium, a layer of air-permeable sealing film is covered on the mouth of the culture bottle, the culture bottle is tightly tied by rubber bands, and then a layer of air-impermeable sealing film is covered on the air-permeable sealing film. When the anther is subjected to high-temperature treatment, the culture bottle is covered by the airtight sealing film, the culture medium cannot lose moisture due to overhigh temperature, immature pollen cannot die due to excessive drying and water loss in the bottle, and when the anther is cultured for