Reagent and method for overcoming self-incompatibility of Chinese cabbage and application

Abstract

The invention belongs to the technical field of brassica plant breeding, and relates to a reagent and method for overcoming self-incompatibility of Chinese cabbage and application. After the Chinese cabbages enter the flowering phase, an EGCG (epigallocatechin gallate) solution is sprayed on the stigmas of the Chinese cabbages, so that the self-incompatibility of the Chinese cabbages can be weakened to different degrees. Experimental results show that after the EGCG is sprayed, the number of seeds obtained from each pod of the self-incompatible Chinese cabbage is increased to 17.8 from original 0. The used reagent EGCG does not pollute the environment, especially the soil environment, has small damage to plants, and can be applied to seed reproduction work of the Chinese cabbage self-incompatible line.

Description

Technical Field

[0001] This invention belongs to the field of Brassica genus breeding technology, specifically relating to a reagent, method and application for overcoming self-incompatibility in Chinese cabbage. Background Technology

[0002] Self-incompatibility is a "self-protection" mechanism evolved in plants, referring to their ability to recognize and inhibit the germination and growth of their own pollen, thereby avoiding self-pollination. This mechanism is very common in plants, especially in cruciferous plants, such as Chinese cabbage, kale, and radish. Self-incompatible lines are ideal materials for producing hybrid Chinese cabbage. By selecting superior self-incompatible lines as parents for hybridization, artificial emasculation can be avoided, improving the purity and quality of hybrid seeds, and thus efficiently producing hybrid seeds. While self-incompatibility has important applications in self-incompatible line seed production, it also brings many difficulties to the efficient propagation of parents.

[0003] Common methods for overcoming self-incompatibility in production include bud-stage pollination and spraying with saline solution. Both have limitations. Bud-stage self-pollination is complex and costly. Spraying with saline solution causes salt stress in plants, affecting growth and development, and can lead to soil salinization. Furthermore, many strongly self-incompatible lines cannot effectively overcome self-incompatibility even with saline solution spraying. Therefore, there is an urgent need to find an efficient and widely applicable method to overcome self-incompatibility in Chinese cabbage. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a reagent to overcome the self-incompatibility of Brassica napus (Chinese cabbage). The technical problem to be solved is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solutions: In a first aspect, the present invention provides an application in overcoming self-incompatibility in Chinese cabbage, wherein the compound is EGCG, and its structural formula is: C 22 H 18 O 11 .

[0006] In the above application, the Chinese cabbage is a self-incompatible Chinese cabbage.

[0007] Secondly, the present invention provides a method for overcoming self-incompatibility in Chinese cabbage, comprising the following steps: spraying the aforementioned compound onto the stigma of self-incompatible Chinese cabbage, and after the stigma dries, pollinating the treated stigma with self-pollination.

[0008] In the above method, the concentration of the compound sprayed is 1 mM-10 mM.

[0009] In the above method, the application is performed 3 times, with an interval of 15 minutes between each application. Each spray is sufficient to observe droplets on the column head.

[0010] The specific method is as follows: 1. One or two days before flowering, use tweezers to remove the already opened flowers of the Chinese cabbage and bag the remaining inflorescences; 2. On the day of pollination, prepare the EGCG working solution by diluting 100 mM EGCG stock solution with 0.0125% Tween-20 (by volume) to obtain 1 mM, 5 mM, and 10 mM EGCG solutions. 3. On a sunny morning between 8:00 and 10:00, spray EGCG solution onto the inflorescence of Chinese cabbage three times, with 15-minute intervals. After each spray, clear droplets should be visible on the stigma. Allow the droplets to dry before continuing to spray. Perform self-pollination 20 minutes after the third spray, and then bag the cabbage after pollination. 4. Observe the germination of pollen tubes 12 hours after pollination. Remove the stigma from the inflorescence and fix it in Carnoy's reagent (anhydrous ethanol: glacial acetic acid = 3:1) until the stigma is completely decolorized. Then soften the stigma with 10 M sodium hydroxide solution in a 42℃ water bath for about 40 min. Finally, stain the stigma in aniline blue solution for more than 30 min and then observe the pollen tube growth using a fluorescence microscope.

[0011] 5. After 50 days, the pollinated pods were removed from the plant and brought back to the laboratory. Tweezers were used to cut them open in the middle to expose the seeds. The pods were then photographed, and the number of seeds in each pod was counted.

[0012] The beneficial effects of this invention are: This invention is applicable to self-incompatible lines of Chinese cabbage. After Chinese cabbage enters the flowering stage, spraying the stigma of the cabbage with EGCG solution can weaken its self-incompatibility to varying degrees. Experimental results show that after spraying EGCG, the number of seeds per pod of self-incompatible Chinese cabbage increased from 0 to 17.8. The EGCG reagent used does not pollute the environment, especially the soil environment, and has minimal harm to plants, making it suitable for propagation of self-incompatible lines of Chinese cabbage. Attached Figure Description

[0013] Figure 1 The images show the growth of pollen tubes in the stigma of Chinese cabbage after EGCG treatment. The left image shows the growth of pollen tubes in the stigma of Chinese cabbage after treatment with different concentrations of EGCG, and the right image shows the number of pollen tubes in the stigma of Chinese cabbage after EGCG treatment.

[0014] Figure 2 The left figure shows the phenotype of Chinese cabbage seeds after EGCG treatment, and the right figure shows the number of seeds in the pods.

[0015] Figure 3 The structure of gallocatechin gallate (EGCG) is shown. Detailed Implementation

[0016] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0017] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0018] The following examples use GraphPad Prism 8.0.1 statistical software to process the data and employ a two-tailed t-test. P <0.01 (**) indicates a significant difference.

[0019] Chinese cabbage 14CR: Described in non-patent literature "Zhang, L. et al. FERONIA receptor kinase-regulated reactive oxygen species mediate self-incompatibility in..." Brassica rapa Curr. Biol. 31, 3004–3016 (2021). This biomaterial is available to the public from Shandong Agricultural University and is intended solely for the purpose of replicating experiments related to this invention; it may not be used for any other purpose.

[0020] Example 1: EGCG treatment method to overcome self-incompatibility in Chinese cabbage 1. Remove weeds: One or two days before flowering, use tweezers to remove the open flowers and pods of the Chinese cabbage, and cover the remaining inflorescences with sulfuric acid paper bags for isolation; 2. Prepare EGCG working solution: EGCG: Purchased from Beijing Solarbio Technology Co., Ltd., item number E8120, purchase website: https: / / www.macklin.cn / info / aboutus.

[0021] Preparation of EGCG stock solution: Dissolve 0.09167 g of EGCG powder in 1 ml of dimethyl sulfoxide, vortex to dissolve, add 1 ml of double-distilled water, and vortex to mix.

[0022] 1 mM EGCG working solution: Dilute 100 mM EGCG stock solution 100 times with water containing 0.0125% Tween-20 by volume to obtain a 1 mM EGCG solution.

[0023] 5 mM EGCG working solution: Dilute 100 mM EGCG stock solution 20 times with water containing 0.0125% Tween-20 by volume to obtain a 5 mM EGCG solution.

[0024] 10 mM EGCG working solution: Dilute 100 mM EGCG stock solution 10 times with water containing 0.0125% Tween-20 by volume to obtain a 10 mM EGCG solution.

[0025] 0 mM EGCG working solution: A 0 mM EGCG solution was obtained using water with a volume fraction of 0.0125% Tween-20.

[0026] 3. On a sunny morning between 8:00 and 10:00, spray the inflorescence of Chinese cabbage with EGCG solution three times, applying each concentration to three cabbages, treating 10 stigmas per cabbage, with 15-minute intervals between applications. After each spray, clear droplets should be visible on the stigmas. Allow the droplets to dry before continuing the spraying process. Self-pollination should be performed 20 minutes after the third spray, followed by bagging the cabbage.

[0027] Example 2: Observation of pollen tube growth by stigma aniline blue staining. After processing as described in Example 1, the following operations are then performed: 1. Twelve hours after pollination, use tweezers to remove the stigma from the inflorescence and fix it in Carnoy's reagent (anhydrous ethanol: glacial acetic acid = 3:1, volume ratio) until the stigma is completely decolorized.

[0028] 2. Wash the decolorized column head with double-distilled water at least 3 times, and then soften the column head with 10 M sodium hydroxide solution in a 42℃ water bath for about 40 minutes.

[0029] 3. After cleaning the column head with double-distilled water to remove sodium hydroxide, place the softened column head in a 0.1% aniline blue solution for staining for at least 30 minutes.

[0030] 4. After staining, use tweezers to remove the stigma, press it into a slide, and observe the number of pollen tubes using a fluorescence microscope with the DAPI channel.

[0031] The results showed that 12 hours after self-pollination in Chinese cabbage, the pollen tubes failed to penetrate the stigma. After treatment with EGCG solution and 12 hours after self-pollination, the pollen tubes of self-pollinated Chinese cabbage successfully penetrated the stigma. Compared to treatments with 1 mM and 10 mM EGCG solutions, treatment with 5 mM EGCG solution resulted in more pollen tubes penetrating the stigma of the Chinese cabbage. Figure 1 ).

[0032] The mock group showed no pod elongation and contained no seeds. Treatment of the stigma of Chinese cabbage with 5 mM EGCG solution resulted in a significantly higher number of seeds per pod compared to other treatments. Therefore, treatment of the stigma of Chinese cabbage with 5 mM EGCG solution can effectively overcome self-incompatibility in Chinese cabbage. Figure 2 ).

[0033] Example 3: Seed Quantity Statistics After processing as described in Example 1, the following operations are then performed: 1. Removing the bag: One week after pollination, the sulfuric acid paper bag can be removed.

[0034] 2. Fifty days after pollination, use scissors to remove the pollinated pods from the plant.

[0035] 3. Apply double-sided tape to the glass slide and fix the pod to the tape. The orientation should be such that either of the two edges of the pod's dorsal or ventral side is facing down.

[0036] 4. Use a dissecting needle to cut along the ridges on the pod and then throw the pod to both sides.

[0037] 5. Observe the number of seeds in the pods using a stereomicroscope and take photos with a camera.

[0038] The mock group consists of cabbage treated with 0 mM EGCG in Example 1.

[0039] The results showed that 50 days after self-pollination, the pods of Chinese cabbage did not elongate and contained no seeds. Treatment of the stigmas with 5 mM EGCG solution significantly increased the number of seeds per pod (17.8 seeds) compared to other treatments (0 seeds in the mock group). Therefore, treatment of the stigmas with 5 mM EGCG solution can effectively overcome self-incompatibility in Chinese cabbage. Figure 2 ).

[0040] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Application in overcoming self-incompatibility in Chinese cabbage, wherein the compound is EGCG, and its structural formula is: C 22 H 18 O 11 .

2. The application according to claim 1, characterized in that, The Chinese cabbage mentioned is a self-incompatible Chinese cabbage.

3. A method for overcoming self-incompatibility in Chinese cabbage, characterized in that, The procedure includes the following steps: spraying the compound described in claim 1 onto the stigma of a self-incompatible Chinese cabbage, and after the stigma dries, pollinating the treated stigma with self-pollination.

4. The method according to claim 3, characterized in that, The concentration of the compound applied is 1 mM-10 mM.

5. The method according to claim 4, characterized in that, The application is performed three times, with an interval of 15 minutes between each application. Each application is considered complete when droplets are observed on the column head.

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