A method for overcoming self-incompatibility in a hybrid european x japanese hazel

Abstract

The application discloses a method for overcoming self-incompatibility of a flat-european hybrid hazel, and uses female flowers of a flat-european hybrid hazel 'Davi' as research materials, selects various chemical methods to carry out experimental research on overcoming self-incompatibility of hazel plants, carries out self-pollination after treating the unpollinated pistil of 'Davi' (using distilled water treated pistil as a control), and uses fluorescence microscopic technology to observe the growth of pollen tubes, so that effective reagents and methods for breaking self-incompatibility and improving pollination compatibility are screened from various chemical reagents, i.e., using 5% acetone solution, 40 mg / L 6-BA solution and 40 mg / L GA solution, carrying out self-pollination after 5 min of treatment, and the efficiency of overcoming self-incompatibility is the highest. The research results provide a theoretical basis for improving the yield of hazelnut and the work efficiency of hybrid breeding, and fill the blank of research on overcoming self-incompatibility in the field of hazel plants at home and abroad.

Description

Technical Field

[0001] This invention belongs to the field of agricultural planting technology, and specifically relates to a method for overcoming the self-incompatibility of European hybrid hazelnuts. Background Technology

[0002] Hazelnuts are a newly emerging economic forest species used for both fruit, oil, and grain, possessing extremely high nutritional and economic value. The Ping-European hybrid hazelnut is an interspecific hybrid offspring of Ping-European and European hazelnuts, with a cultivated area of ​​approximately 2 million mu (about 133,333 hectares) nationwide, making it a major cultivated hazelnut species in my country. Hazelnut flowers are unisexual, with male and female flowers on the same plant but separate, exhibiting self-incompatibility or cross-pollination incompatibility (Mehlenbacher & Thompson, 1988). Overcoming self-incompatibility and achieving successful pollination and fertilization are crucial prerequisites for nut production and hybridization breeding. Given the self-incompatibility of hazelnuts, production often employs mixed planting of multiple varieties, with each variety acting as a pollinator; however, in hybridization breeding, due to self-incompatibility, certain hybrid combinations of specific parents cannot be achieved.

[0003] Self-incompatibility is a widespread reproductive isolation mechanism that prevents self-pollination and promotes cross-pollination by recognizing both self-pollination and cross-pollination, thereby maintaining the genetic diversity of species. Self-pollination refers not only to pollen from the same plant but also to pollen from all plants with the same genotype at the S locus (the gene locus controlling self-incompatibility in plants). It has been reported that approximately 40% of species in over 100 plant families exhibit self-incompatibility. Based on relevant research reports both domestically and internationally, various methods have been applied to overcome plant self-incompatibility, which can be categorized into four types: biological methods, physical methods, chemical methods, and molecular methods. While the first two methods have some effectiveness, they are cumbersome, time-consuming, and labor-intensive, and can easily damage the pistil to some extent. Researchers both domestically and internationally primarily focus on applying chemical and molecular methods to overcome self-incompatibility.

[0004] In view of the above shortcomings, this invention uses the female flowers of the European hybrid hazelnut 'Davi' as research material, employs chemical methods to treat the pistils for self-pollination, and uses fluorescence microscopy to observe the growth of pollen tubes. It screens out effective reagents and methods to overcome self-incompatibility and improve pollination compatibility, providing a theoretical basis for improving hazelnut nut yield and hybridization breeding efficiency. Summary of the Invention

[0005] Currently, there are no reports on overcoming self-incompatibility in the field of hazelnut research, both domestically and internationally. Based on this, this invention uses the female flowers of the European hybrid hazelnut 'Davi' as research material, and selects various chemical methods to conduct experimental research on overcoming self-incompatibility in hazelnuts. After treating the unpollinated pistils of 'Davi' (with distilled water-treated pistils as a control), self-pollination was performed. The growth of pollen tubes was observed using fluorescence microscopy. Effective reagents and methods for breaking self-incompatibility and improving pollination compatibility were screened from various chemical reagents, providing a theoretical basis for improving hazelnut nut yield and the efficiency of hybridization breeding.

[0006] This invention is achieved using the following technical means:

[0007] A method for overcoming self-incompatibility in European hybrid hazelnuts includes the following steps:

[0008] (1) Treatment method: Before pollination, invert the female flower of 'Davimid' and dip it lightly in the corresponding reagent for a few seconds and then shake it slightly to prevent droplets from forming. After the solution evaporates, 'Davimid' self-pollination is carried out.

[0009] (2) Self-pollination: After a certain period of time, the female flowers were placed upright and self-pollinated. The pollinated female flowers were placed in a petri dish with double layers of wet filter paper, covered to prevent moisture evaporation, and stored at room temperature. After 24 hours of pollination, the growth status of the pollen tubes was observed using fluorescence microscopy.

[0010] Further, the corresponding reagents in step (1) are selected from acetone, 6-BA solution and GA solution.

[0011] Furthermore, the concentration of the acetone solution is 5%; the concentration of the 6-BA solution is 40 mg / L; and the concentration of the GA solution is 40 mg / L.

[0012] Furthermore, the certain time mentioned in step (2) is 5 minutes.

[0013] Furthermore, to ensure the growth and development of the pollinated female flowers, all operations were carried out at room temperature (25°C, 60% relative humidity).

[0014] The beneficial effects of this invention are as follows:

[0015] This invention uses the female flowers of the European hybrid hazelnut 'Davi' as research material. Multiple chemical methods were selected to conduct experimental research on overcoming self-incompatibility in hazelnut plants. Unpollinated pistils of 'Davi' were treated (with pistils treated with distilled water as a control) before self-pollination. Pollen tube growth was observed using fluorescence microscopy. Effective reagents and methods for overcoming self-incompatibility and improving pollination compatibility were screened from various chemical reagents. Specifically, treatment with 5% acetone solution, 40 mg / L 6-BA solution, and 40 mg / L GA solution for 5 minutes before self-pollination showed the highest efficiency in overcoming hybridization incompatibility. The above research results provide a theoretical basis for improving hazelnut nut yield and the efficiency of hybridization breeding, filling a gap in research on overcoming self-incompatibility in hazelnut plants both domestically and internationally. Attached Figure Description

[0016] Figure 1 The images show the growth of self-pollination pollen tubes after treatment with different reagents. (a), (b), and (c) represent the growth of pollen tubes after treatment with 6-BA, acetone, and GA, respectively; (d), (e), and (f) represent the growth of pollen tubes after treatment with distilled water. Detailed Implementation

[0017] The preferred embodiments of the present invention are described below. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0018] Example 1

[0019] A method for overcoming self-incompatibility in European hybrid hazelnuts includes the following steps:

[0020] (1) Materials: Female flowers and fresh pollen of the European hybrid hazelnut 'Davi' were used as materials. The exogenous chemical reagents and their treatment concentrations to overcome self-incompatibility are shown in Table 1.

[0021] Table 1. Reagents and their treatment concentrations

[0022]

[0023] (2) Method

[0024] ① Treatment method: 10 female flowers of 'Daviria' were selected as the research object for each treatment; before pollination, the female flowers were inverted and lightly dipped in the corresponding reagent for a few seconds and then gently shaken to prevent droplet formation. After the solution evaporated, 'Daviria' was self-pollinated, and distilled water treatment was used as a control.

[0025] ② Self-pollination: Place the female flower upright and use a cotton swab to evenly spread fresh 'Davi' pollen onto the pistil. After pollination, place 10 female flowers in a petri dish with double layers of moist filter paper, cover, and weigh them down with an appropriate weight to prevent moisture evaporation. Store at room temperature. 24 hours after pollination, observe the growth of pollen tubes using fluorescence microscopy.

[0026] ③ Fluorescence microscopy observation: Use tweezers and a dissecting needle to separate the pistils from the female flower buds and distribute them evenly on a glass slide (there should be no overlap or overlap between the pistils), and count the number of pistils; use a dropper to draw a small amount of aniline blue staining solution (pH 8.0-9.0) and drop it onto the glass slide, cover it with a coverslip, and gently tap the pistil area with the blunt end of tweezers or a dissecting needle or other similar instrument to make it flat between the glass slide and the coverslip, and fully contact the aniline blue solution; wipe off the excess aniline blue solution with a paper towel, and observe the pollen tube growth under a fluorescence microscope.

[0027] ④ Pollen tube penetration rate: Hazelnut female flowers consist of a cluster of pistils, and the entire surface of the pistil is capable of receiving pollen. Pollination compatibility is indicated by any single pollen tube penetrating the pistil surface and entering the style channel tissue. The pollen tube penetration rate is used to evaluate the effectiveness of different reagents in overcoming self-incompatibility. Pollen tube penetration rate = (Number of pistils with pollen tubes penetrating the pistil surface / Total number of pistils treated) × 100%.

[0028] ⑤ Screening of suitable pollination time after reagent treatment: Four pollination time points were designed after reagent treatment of the pistils (immediate pollination, pollination after 5 minutes, pollination after 10 minutes, and pollination after 15 minutes) to screen the optimal pollination time after reagent application. Five female flowers were selected for treatment at each pollination time point. To ensure the growth and development of the pollinated female flowers, all operations were carried out at room temperature (temperature 25℃, relative humidity 60%).

[0029] (3) Results and Analysis

[0030] ① Comparison of the effects of different reagents on overcoming self-incompatibility:

[0031] The experimental results show that all selected reagents can overcome the self-incompatibility of 'Davidi' to some extent, but the effects of different reagents on overcoming the self-incompatibility of hazelnut plants vary greatly. Table 2 shows that all seven reagents selected in this invention achieved significant effects compared to the distilled water treatment. Looking at the treatment effects of these seven reagents alone, GA, 6-BA, and acetone are significantly better than sodium chloride + boric acid, 'Liaoning Hazelnut No. 7' pollen extract, ethanol, and ammonium sulfate. Among the three reagents with the most significant effects, based on pollen tube penetration rate, 6-BA has the most obvious effect, followed by acetone, and GA ranks third. Although the other reagents show significant differences in treatment effects compared to the control, the pollen tube penetration rate indicates that the treatment effects of these reagents are not ideal. The pollen tube growth of 'Davidi' after self-pollination following treatment with 6-BA, acetone, and GA is shown in the figure. Figure 1 As shown in a, b, and c, the control samples treated with distilled water are as follows: Figure 1 As shown in figures d, e, and f, it is clear from the figures that after treatment with these three reagents, the pollen tubes of self-pollinating plants penetrated the stigma surface and grew parallel downwards along the style channel tissue; while in the control sample treated with distilled water, the pollen tubes were twisted and coiled, failing to penetrate the stigma surface. Therefore, 6-BA, acetone, and GA are considered to be relatively effective reagents for overcoming self-incompatibility in hazelnut plants.

[0032] Table 2 Comparison of the effects of different reagents on overcoming the self-incompatibility of the European hybrid hazelnut 'Davi'

[0033]

[0034] ② The effect of different reagent concentrations on overcoming self-incompatibility:

[0035] The breakthrough effects of different concentrations of the same reagent and the breakthrough effects of all 21 reagent treatments were compared (Table 3). Within the 6-BA treatment group, the breakthrough rate ranged from 48.958% to 58.852%, with a concentration of 40 mg / L showing the most significant effect and the highest breakthrough rate. Within the acetone treatment group, the breakthrough rate ranged from 45.455% to 56.744%, with a concentration of 5% showing the most significant effect and the highest breakthrough rate. Within the GA treatment group, the breakthrough rate ranged from 48.980% to 53.704%, with a concentration of 40 mg / L showing the most significant effect and the highest breakthrough rate. Within the ethanol treatment group, the breakthrough rate ranged from 24.615% to 31.937%. The 50% concentration showed the most significant effect and the highest penetration rate. Within the ammonium sulfate treatment group, the penetration rate ranged from 32.174% to 48.168%, with a 5% concentration showing the most significant effect and the highest penetration rate. In the sodium chloride + boric acid treatment group, the penetration rate ranged from 29.817% to 38.889%, with a 10% + 1% concentration showing the most significant effect and the highest penetration rate. Within the 'Liaoning Hazelnut 7' pollen extract treatment group, the penetration rate ranged from 24.107% to 33.684%, with a 1:15 concentration showing the most significant effect and the highest penetration rate. Comparison of all 21 reagent treatments showed that 40 mg / L 6-BA and 5% acetone were the most effective in overcoming self-incompatibility; 40 mg / L GA also showed good results. Therefore, the concentration of the reagent has a significant impact on the effectiveness of overcoming self-incompatibility. Inappropriate concentrations can often lead to significant deviations in experimental results, masking the true effect of the reagent. Therefore, after initially selecting suitable reagents, preparing appropriate reagent concentrations is essential to achieve the best results in overcoming the problem.

[0036] ③ The effect of assisted pollination time on overcoming self-incompatibility:

[0037] Based on the above experimental results, 40 mg / L 6-BA and 5% acetone were selected to determine the optimal pollination time after reagent treatment. According to the experimental results (Table 4), it can be seen that for both 5% acetone and 40 mg / L 6-BA, immediate pollination after reagent treatment resulted in the lowest penetration rates, at 19.167% and 25.556%, respectively. The most significant effect was observed when pollination was performed 5 minutes after treatment, with the penetration rates reaching their maximum values ​​of 54.545% and 54.386%, respectively. As the pollination time after reagent treatment increased, the penetration rate gradually decreased, and the treatment effect gradually weakened. Therefore, pollination 5 minutes after reagent treatment ensures that the reagent's effect in overcoming self-incompatibility is optimal.

[0038] Table 3. Effects of different reagent concentrations on overcoming the self-incompatibility of the European hybrid hazelnut 'Davi'

[0039]

[0040]

[0041] Table 4. Effect of auxiliary pollination time on overcoming self-incompatibility in the European hybrid hazelnut 'Davi'

[0042]

[0043] To explore effective methods for overcoming self-incompatibility in the European hybrid hazelnut 'Davi', this invention selected seven reagents and configured them with different concentration gradients. The pollen tube penetration rate was used as the standard to determine the effectiveness of different reagents in overcoming self-incompatibility. Experimental results showed that acetone, GA, and 6-BA were relatively effective in overcoming self-incompatibility, with pollen tube penetration rates exceeding 50% after solution treatment. The optimal concentrations for the three reagents were 5% acetone solution, 40 mg / L 6-BA solution, and 40 mg / L GA solution. Treating with the chemical agents for 5 minutes before self-pollination resulted in the highest efficiency in overcoming hybridization incompatibility.

[0044] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for overcoming self-incompatibility in European hybrid hazelnuts, comprising: (1) Pretreatment: Before pollination, the female flowers of 'Davimid' were dipped in the corresponding reagent for pretreatment. The female flowers of 'Davimid' were inverted, dipped in the corresponding reagent for a few seconds and then shaken slightly to prevent droplet formation and wait for the solution to evaporate. (2) Self-pollination: After a certain period of time, the female flowers of 'Davi' were subjected to self-pollination; among which: The corresponding reagents are selected from: 5% acetone, 40 mg / L 6-BA solution, and 40 mg / L GA solution; The specified time is 5 minutes.

2. The method according to claim 1, wherein: Step (2) of self-pollination includes: After a certain period of time, the female flowers were placed upright and self-pollinated. The pollinated female flowers were then placed in a petri dish with double layers of moist filter paper, covered to prevent moisture evaporation, and stored at room temperature. 24 hours after pollination, the growth of pollen tubes was observed using fluorescence microscopy.

3. The method according to claim 1, wherein: Both the pretreatment and self-pollination need to be carried out at a temperature of 25°C and a relative humidity of 60%.

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