A method for overcoming self-incompatibility reaction in cruciferous plants

Abstract

The present invention discloses a method for overcoming self-incompatibility reaction in cruciferous plants, which relates to the field of plant biotechnology. This method is to treat the stigma tissues of self-incompatible Arabidopsis thaliana and Chinese cabbage with the autophagy process inhibitor wortmannin, then perform self-pollination on the treated stigma tissues, and finally obtain the self-crossed seeds of self-incompatible plants. The present invention has the following advantages and positive effects: ① This method uses the autophagy process inhibitor wortmannin to treat the stigmas of self-incompatible Arabidopsis thaliana and Chinese cabbage for the first time, which can effectively overcome their self-incompatibility and obtain self-crossed seeds. ② This method uses a single agent for treatment, with simple operation and stable effect, and does not require the aid of special tool equipment. ③ Using this method to overcome the self-incompatibility reaction has a stable effect in different species of cruciferous plants, providing a theoretical and practical basis for expanding the application range of self-incompatible lines in heterosis utilization.

Description

Technical Field

[0001] The present invention relates to the field of plant biotechnology, and particularly to a method for overcoming the self-incompatibility reaction of cruciferous plants. Specifically, wortmannin is used to treat the stigmas of self-incompatible Arabidopsis thaliana and Chinese cabbage, and then the self-seeds of self-incompatible plants are obtained. Background Art

[0002] Self-incompatibility refers to the phenomenon that flowering plants can produce normal male and female gametes, but self-pollination cannot occur or seeds cannot be set after pollination. Most cruciferous plants have self-incompatibility. The utilization of heterosis is an important way to improve crop yield and quality. Self-incompatibility is one of the important ways to utilize heterosis. Compared with other ways of utilizing heterosis, self-incompatible lines have the advantages of being easy to breed, having no adverse cytoplasmic effects, and having a high hybrid rate. However, due to the existence of self-incompatibility, the self-seed propagation of self-incompatible lines is an urgent problem to be solved in current breeding.

[0003] Currently, the main methods for propagating self-incompatible lines include bud pollination method, CO 2 treatment method, spraying NaCl solution method, etc. Among them, the bud pollination method is to pollinate and propagate self-incompatible lines by removing buds 2-5 days before flowering. At this time, the self-incompatibility recognition genes of Brassica plants have not been highly expressed, and self-pollen cannot be recognized by the stigma. However, this method is laborious and time-consuming, with high production costs and strict technical requirements, restricting the seed propagation efficiency. Treating self-incompatible plants with high-concentration (4%) CO 2 during the flowering period can effectively overcome their self-incompatibility. However, the treatment effects of this method vary greatly among different varieties and cannot be widely applied. Spraying self-incompatible plants with an appropriate concentration (3-5%) of NaCl solution during the flowering period can also effectively propagate self-incompatible lines, but among different cruciferous species, the required NaCl solution concentration varies greatly and sometimes other reagents such as boric acid need to be added for assistance.

[0004] In recent years, researchers have found that the autophagy process plays an important role in the self-incompatibility reaction of cruciferous plants, and knocking out the key genes of the autophagy process can break the self-incompatibility reaction. Using the autophagy process inhibitor wortmannin to treat the stigma to overcome the self-incompatibility reaction of cruciferous plants and obtain self-seeds of self-incompatible lines has important application value. Summary of the Invention

[0005] The object of the present invention is to overcome the disadvantages and deficiencies existing in the prior art and provide a method for overcoming the self-incompatibility reaction of cruciferous plants; another object of the present invention is to obtain a large number of self-seeds of self-incompatible lines in the utilization of heterosis by this method.

[0006] The technical solution of the present invention is as follows:

[0007] Treat the stigma tissues of self-incompatible Arabidopsis thaliana and Chinese cabbage with wortmannin, an inhibitor of the autophagy process, then perform self-pollination on the treated stigma tissues, and finally obtain self-crossed seeds of self-incompatible plants.

[0008] Specifically, this method includes the following steps:

[0009] ① On the day before flowering, emasculate the flower buds of self-incompatible Arabidopsis thaliana or Chinese cabbage;

[0010] ② The next day, prepare the wortmannin working solution for Arabidopsis thaliana: Dig an appropriate amount of lanolin stored at 4°C into a 1.5 mL centrifuge tube, place it in a 50°C water bath for several minutes until completely melted, mix 1 mM wortmannin stock solution with lanolin in a volume ratio of 1:2 evenly to prepare a wortmannin working solution with a final concentration of 0.33 mM;

[0011] ③ Prepare the wortmannin working solution for Chinese cabbage: Add 1 μL of 10 mM wortmannin stock solution to 19 μL of ultrapure water to prepare a wortmannin working solution with a final concentration of 0.5 mM;

[0012] ④ Treatment of Arabidopsis thaliana with wortmannin: Under a stereomicroscope, dip an anatomical needle into an appropriate amount of the wortmannin working solution for Arabidopsis thaliana, and carefully apply it around the style of the emasculated pistil for 3 hours;

[0013] ⑤ Treatment of Chinese cabbage with wortmannin: Aspirate 1 μL of the wortmannin working solution for Chinese cabbage and drop it onto the stigma of the emasculated Chinese cabbage. After 2 hours of treatment, carefully blot the residual reagent on the stigma until the papilla cells are completely dry and then proceed to the next step;

[0014] ⑥ Pollination: Take the anthers of self-incompatible Arabidopsis thaliana or Chinese cabbage that are shedding pollen, and perform artificial pollination on the stigmas of the treated Arabidopsis thaliana or Chinese cabbage;

[0015] ⑦ Aniline blue staining to observe pollen tube germination: Remove the pistils of self-incompatible Arabidopsis thaliana and Chinese cabbage 24 hours after pollination and place them in a fixing solution for fixation, and then soften the pistils in a NaOH solution; Place the softened pistils in an aniline blue solution for staining; Finally, observe with a fluorescence microscope;

[0016] ⑧ Pollinate Arabidopsis thaliana and harvest self-crossed seeds.

[0017] The present invention has the following advantages and positive effects:

[0018] ① This method first uses wortmannin, an inhibitor of the autophagy process, to treat the stigmas of self-incompatible Arabidopsis thaliana and Chinese cabbage, and effectively overcomes their self-incompatibility to obtain self-crossed seeds.

[0019] ② This method uses a single reagent for treatment, which is simple to operate, has stable effects, and does not require the aid of special tool equipment.

[0020] ③ Using this method to overcome self-incompatibility reactions has stable effects in different species of cruciferous plants, providing a theoretical and practical basis for expanding the application scope of self-incompatible lines in heterosis utilization. Description of the Drawings

[0021] Figure 1 It is a schematic diagram of the wortmannin treatment method for Arabidopsis thaliana.

[0022] Figure 2 It is a picture of the pollen tube germination on the stigma after wortmannin treatment of Arabidopsis thaliana and Chinese cabbage. Among them:

[0023] 2a shows the growth of pollen tubes on the papilla cells of the stigma of self-incompatible Arabidopsis thaliana without treatment (left) and after wortmannin treatment (right); scale bar: 100 μm.

[0024] 2b shows the growth of pollen tubes on the papilla cells of the stigma of Chinese cabbage without treatment (left) and after wortmannin treatment (right); scale bar: 200 μm.

[0025] Figure 3 It is a picture of the seed setting situation of self-incompatible Arabidopsis thaliana without treatment (top) and after wortmannin treatment (bottom); scale bar: 2 mm. Detailed Embodiments

[0026] The following is a detailed description in conjunction with the drawings and embodiments:

[0027] Unless otherwise specified, the following test methods are all conventional methods and techniques in this technical field, and the reagents and consumables used can be obtained through commercial channels.

[0028] I. Further

[0029] 1. For the wortmannin working solution of Arabidopsis thaliana in step ②, when the room temperature is relatively low, the wortmannin stock solution (concentration) and lanolin can be mixed evenly at a volume ratio of 1:2 in a 45 °C water bath.

[0030] 2. In step ④, when applying the wortmannin working solution of Arabidopsis thaliana around the pistil style of the emasculated flower, care should be taken to avoid the working solution from contacting the papilla cells of the stigma.

[0031] II. Embodiment

[0032] 1. Embodiment 1, Preparation of wortmannin reagent and treatment of self-incompatible lines of Arabidopsis thaliana and Chinese cabbage

[0033] 1) Emasculation: One day before anthesis, manually emasculate the flower buds of self-incompatible Arabidopsis thaliana and Chinese cabbage at flower development stage 12 (stage 12). Use forceps to remove tissues such as sepals, petals, and stamens, so that the pistil is completely exposed, and bag the emasculated pistils for isolation.

[0034] 2) Preparation of wortmannin working solution for Arabidopsis thaliana: On the day of treatment, take an appropriate amount of solid lanolin stored at 4°C into a 1.5 mL centrifuge tube, place it in a 50°C water bath for several minutes until completely melted, and stir and mix the wortmannin stock solution (1 mM) and lanolin thoroughly at a volume ratio of 1:2 to prepare a wortmannin working solution with a final concentration of 0.33 mM.

[0035] 3) Preparation of wortmannin working solution for Chinese cabbage: On the day of treatment, add 1 μL of wortmannin stock solution (10 mM) to 19 μL of ultrapure water to prepare a wortmannin working solution with a final concentration of 0.5 mM.

[0036] 4) Treatment of Arabidopsis thaliana with wortmannin: Under a stereomicroscope, use a dissecting needle to dip an appropriate amount of the wortmannin working solution for Arabidopsis thaliana and carefully apply it around the style of the emasculated pistil, taking care not to let the working solution touch the papilla cells. Treat for 3 hours, and the treatment method is as Figure 1 shown.

[0037] 5) Treatment of Chinese cabbage with wortmannin: Aspirate 1 μL of the wortmannin working solution for Chinese cabbage and drop it onto the stigma of the emasculated Chinese cabbage. After 2 hours of treatment, carefully blot the residual reagent on the stigma until the papilla cells are completely dry before proceeding to the next step.

[0038] 2. Example 2, Pollination and aniline blue staining of self-incompatible Arabidopsis thaliana and Chinese cabbage after wortmannin treatment

[0039] 1) Pollination: Take the anthers of self-incompatible Arabidopsis thaliana or Chinese cabbage that are shedding pollen and perform artificial pollination on the stigmas of the treated Arabidopsis thaliana or Chinese cabbage.

[0040] 2) Aniline blue staining:

[0041] a. Use forceps to remove Arabidopsis thaliana and Chinese cabbage 24 hours after pollination from the flower stalk and soak them in Carnoy's fixative (glacial acetic acid: absolute ethanol = 1:2) for about 2 hours of fixation;

[0042] b. Take out the fixed pistils and rinse them with ultrapure water more than 3 times, place them in 1 M NaOH solution, and soften them in a 60°C water bath for 1 hour;

[0043] c. Take out the softened pistils and place them in 0.1% aniline blue staining solution (dissolved in 0.1 M K 3 PO 4in the buffer solution, stain for 2.5 hours in the dark;

[0044] 3. Example 3, observe the germination of pollen tubes on the stigma under a fluorescence microscope

[0045] 1) Preparation of sample slides: Use forceps to take out the stained pistils and place them on a glass slide with distilled water dropped on it, and gently press the cover glass.

[0046] 2) Microscopic observation: Place the slide upside down on the stage of an inverted fluorescence microscope, move the pistil to the center of the field of view, and observe the germination of pollen tubes on the stigma under the excitation of a 405 nm wavelength; the aniline blue staining results of the self-incompatible Arabidopsis thaliana treated with wortmannin are as Figure 2 shown in a; the aniline blue staining results of the self-incompatible Chinese cabbage treated with wortmannin are as Figure 2 shown in b.

[0047] Example 4, harvesting of self-incompatible Arabidopsis thaliana self-seeds

[0048] 1) Pollination: Take the anthers of self-incompatible Arabidopsis thaliana that are shedding pollen and perform artificial pollination on the stigmas of the treated Arabidopsis thaliana.

[0049] 2) Harvesting of self-seeds:

[0050] a. Two weeks after pollination, use forceps to remove the siliques and place them in a 75% ethanol solution for decolorization for one week.

[0051] b. Use forceps to take out the decolorized siliques and place them on a glass slide, place the glass slide on the stage of a stereomicroscope, move the siliques to the center of the field of view, and observe the development of the siliques in bright field; the development of the siliques is as Figure 3 shown.

Claims

1. A method for overcoming the self-incompatibility reaction of cruciferous plants, characterized in that: The stigma tissues of self-incompatible Arabidopsis and Chinese cabbage were treated with wortmannin, an inhibitor of the autophagy process, and then the treated stigma tissues were self-pollinated to obtain self-incompatible plants. ① The self-incompatible Arabidopsis or Chinese cabbage buds were emasculated one day before flowering; ② The next day, prepare the Arabidopsis wortmannin working solution: scoop out an appropriate amount of lanolin stored at 4°C into a 1.5 mL centrifuge tube, place it in a 50°C water bath for several minutes until it is completely melted, mix 1 mM wortmannin stock solution and lanolin in a volume ratio of 1:2 to prepare a wortmannin working solution with a final concentration of 0.33 mM; ③ Prepare Chinese cabbage wortmannin working solution: add 1 μL of wortmannin stock solution 10 mM to 19 μL of ultrapure water to prepare a wortmannin working solution with a final concentration of 0.5 mM; ④ Treatment of Arabidopsis wortmannin: Under a stereo microscope, use a dissecting needle to dip an appropriate amount of Arabidopsis wortmannin working solution, carefully apply it around the style of the emasculated pistil, and treat for 3 hours; ⑤ Treatment of Chinese cabbage with wortmannin: Pipette 1 μL of Chinese cabbage wortmannin working solution and drop it onto the stigma of the emasculated Chinese cabbage. After 2 hours of treatment, carefully dry the residual reagent on the stigma until the papillary cells are completely dry before proceeding to the next step; ⑥ Pollination: Take the anthers of self-incompatible Arabidopsis or Chinese cabbage that are shedding pollen, and artificially pollinate the treated stigmas of Arabidopsis or Chinese cabbage; ⑦ Observation of pollen tube germination by aniline blue staining: 24 hours after pollination, the pistils of self-incompatible Arabidopsis and Chinese cabbage were removed and fixed in fixative, then the pistils were placed in NaOH solution, and the softened pistils were stained with aniline blue solution, and finally observed under a fluorescence microscope; ⑧ Pollinate Arabidopsis thaliana or Chinese cabbage to harvest self-fertilized seeds.

2. The method for overcoming the self-incompatibility reaction of cruciferous plants according to claim 1, characterized in that: The Arabidopsis wortmannin working solution in step ② can be prepared by mixing the wortmannin mother solution and lanolin in a volume ratio of 1:2 in a 45° C. water bath when the room temperature is low.

3. The method for overcoming the self-incompatibility reaction of cruciferous plants according to claim 1, characterized in that: In the step ④, when applying the Arabidopsis thaliana wortmannin working solution around the style of the emasculated pistil, the working solution should be prevented from contacting the stigma papilla cells.

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