Black poplar and white poplar distant hybridization obstacle breakthrough method based on sodium benzoate treatment

Abstract

The invention relates to the technical field of forest tree genetic breeding and plant reproduction regulation and control, and particularly discloses a black poplar and white poplar distant hybridization obstacle breakthrough method based on sodium benzoate treatment. According to the method, female flowers of black poplar are taken as a treatment object, a sodium benzoate aqueous solution with the final concentration of 5-15 mM (preferably 10 mM) is used for carrying out dipping treatment on the female flowers, and then poplar pollen pollination is carried out. After pollination, fixing, softening and aniline blue dyeing are carried out, and pollen germination and pollen tube growth conditions are observed. Experimental results show that the method can significantly promote the growth of the poplar pollen on the black poplar stigma, improve the ability of a pollen tube to penetrate through the stigma, and realize fertilization and hybrid embryo formation, thereby effectively breaking through the distant hybridization obstacle between different branches of the poplar. The method disclosed by the invention is simple and convenient to operate, low in cost and good in repeatability, provides an efficient and feasible technical means for distant hybridization breeding of poplars, and has a good popularization and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of forest tree genetics and breeding and plant reproductive regulation technology, specifically involving a method for using sodium benzoate to regulate pollen-stigma interaction, thereby promoting the germination of poplar pollen on the stigma of black poplar and improving the compatibility of distant hybridization. Background Technology

[0002] Poplar (Populus) is an important fast-growing timber species widely cultivated worldwide, with significant applications in ecological restoration, timber production, and biomass energy. Integrating superior traits from different species or groups through distant hybridization is a crucial approach to poplar genetic improvement.

[0003] However, significant barriers to distant hybridization are prevalent among different branches of the Populus genus (such as the black poplar and white poplar), mainly manifested as inhibited pollen germination or the inability of pollen tubes to penetrate the style tissue, leading to fertilization failure. This pollen-stigma incompatibility severely limits the introduction and utilization of desirable traits.

[0004] In plant distant hybridization research, existing techniques typically attempt to promote hybridization compatibility through pollen pretreatment, hormone treatment, or embryo rescue. However, these methods are mainly applicable to herbaceous crops such as cruciferous plants or post-fertilization obstacles. Currently, there is a lack of effective intervention methods for distant hybridization of forest trees, particularly for the problems of early pollen growth and pollen tube elongation exhibited between black poplar and white poplar species.

[0005] Currently, there are no clearly reported techniques that can significantly promote the germination and growth of white poplar pollen on the stigma of black poplar through simple treatments, thus facilitating fertilization. Therefore, developing a technique that can directly act on the stigma interface to improve heterologous pollen germination and pollen tube growth is of great significance for overcoming the limitations of distant hybridization in poplar. Summary of the Invention

[0006] The purpose of this invention is to provide a method for promoting the growth of white poplar pollen on the stigma of black poplar and the penetration of pollen tubes through the style of black poplar using sodium benzoate, thereby improving the success rate of distant hybridization in poplar trees. The method includes the following steps:

[0007] Step 1: Select mature female inflorescences of black poplar;

[0008] Step 2: Prepare sodium benzoate aqueous solution: The concentration of the sodium benzoate solution is 5 mM-15 mM, preferably 10 mM;

[0009] Step 3: Immerse the female flowers of Populus tomentosa in the sodium benzoate solution for treatment: the stigma treatment time is approximately 30 seconds;

[0010] Step 4: Immediately after treatment, pollinate the poplar flowers;

[0011] Step 5: After pollination for a certain period of time, fix the female flowers in Carnott's fixative.

[0012] Step 6: Soften the fixed female flowers with sodium hydroxide solution, and then stain them with aniline blue solution.

[0013] Step 7: Prepare slides of the stained female flowers and observe the pollination status using a microscope (OLYMPUS BX43F, TOKYO, JAPAN).

[0014] The beneficial effects of this invention are:

[0015] Compared with the prior art, the present invention has the following advantages:

[0016] 1. Under untreated conditions, white poplar pollen germinates slowly on the stigma of black poplar and the pollen tube cannot grow normally through the style. However, the present invention can significantly promote pollen germination and pollen tube elongation by short-term treatment with sodium benzoate, thus achieving the fertilization process.

[0017] 2. The method is simple and requires only a short treatment time to significantly improve the success rate of distant hybridization;

[0018] 3. No gene editing or complex tissue culture techniques are required; the operation is convenient and the cost is low.

[0019] 4. This invention provides for the first time a stable and reproducible technical means to overcome the barrier of distant hybridization between black poplar and white poplar;

[0020] 5. This provides a feasible technical means for distant hybridization breeding of poplar trees, which has important theoretical significance and application value. Attached Figure Description

[0021] Figure 1 Schematic diagram of the inflorescence and floral structure of black poplar and white poplar;

[0022] Figure 2 The effect of sodium benzoate treatment on the germination of Populus tomentosa pollen on the stigma of Populus nigra.

[0023] Figure 3 The effect of sodium benzoate treatment on the growth of pollen tubes in the style of Populus tomentosa;

[0024] Figure 4 This describes the ovary development and hybrid embryo formation after sodium benzoate treatment.

[0025] Blue light is used to observe pollen attachment, while ultraviolet light is used to observe pollen tube growth. Detailed Implementation

[0026] The present invention will be further described below with reference to the embodiments, but the present invention is not limited to the following embodiments.

[0027] 1. Plant materials

[0028] Black poplar (Populus × xiaohei) and white poplar (Populus davidiana) were planted in the experimental forest farm of Northeast Forestry University. Mature pollen was collected during the flowering period, screened, and stored at 4℃ for later use. Mature female inflorescences of black poplar were selected for pollination experiments.

[0029] 2. Preparation of sodium benzoate solution

[0030] Sodium benzoate was dissolved in distilled water to prepare a mother solution, which was then stored at low temperature for later use. Before use, the mother solution was diluted to working concentrations of 5 mM, 10 mM, and 15 mM.

[0031] 3. Stigmata treatment and pollination

[0032] Mature female poplar flowers were immersed in sodium benzoate working solution for 30 seconds. This treatment time is the effective treatment condition verified by this invention, which can stably promote heterologous pollen germination and pollen tube growth. Poplar pollination was performed immediately after removal. The treated inflorescences were then cultured in a greenhouse.

[0033] 4. Pollen germination and pollen tube observation

[0034] After pollination, the female flowers were fixed in Carnoy's fixative for a certain period of time, softened with sodium hydroxide, and then stained with aniline blue. The pollen germination and pollen tube growth were observed using a fluorescence microscope.

[0035] 5. Explanation of Experimental Results

[0036] Experimental results showed that, compared with the untreated group, sodium benzoate treatment significantly improved the growth of poplar pollen on the stigma of black poplar and promoted pollen tube penetration through the style to the ovary. Simultaneously, the treated group was able to form normally developing hybrid embryos, while the stigma of the untreated group rapidly deteriorated after pollination, failing to complete the fertilization process. This indicates that sodium benzoate treatment can effectively promote the germination of poplar pollen on the stigma of black poplar and pollen tube growth, thereby overcoming the barrier to distant hybridization.

[0037] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. All modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An application of sodium benzoate to promote the germination and growth of poplar pollen on female poplar flowers and improve the compatibility of distant hybridization, characterized in that, Includes the following steps: (1) Treat the female flowers of black poplar with sodium benzoate solution before pollination; (2) After treatment, conduct pollination with poplar pollen; The final concentration of the sodium benzoate solution is 5 mM-15 mM.

2. The application according to claim 1, characterized in that: The final concentration of the sodium benzoate solution is preferably 10 mM.

3. The application according to claim 1 or 2, characterized in that: The treatment method is immersion treatment.

4. The application according to claim 3, characterized in that: The female flower immersion treatment time is approximately 30 seconds.

5. The application according to any one of claims 1–4, characterized in that: The pollination refers to heterologous pollination of female flowers of Populus tomentosa by Populus tomentosa.

6. The application according to any one of claims 1–5, characterized in that: The method can promote pollen tubes to pass through the style and enter the ovary.

7. The application according to any one of claims 1–6, characterized in that: The method can improve the fertilization success rate and hybrid embryo formation rate after distant pollination.

8. The method according to any one of claims 1–7, characterized in that: The black poplar is Populus × xiaohei, and the white poplar is Populus davidiana.

9. The method according to any one of claims 1–8, characterized in that: The treatment promotes pollen tube growth by regulating the state of the pollen-stigma interaction.

Images