An agent that overcomes distant hybridization incompatibility in plants of the family rutaceae and methods of use thereof

Abstract

The application discloses a reagent for overcoming distant hybridization incompatibility of plants in the Rutaceae family and a use method thereof. 2+ The reagent is EDTA or EGTA solution which is sprayed on the stigma of the plants in the Rutaceae family, so that the Ca content of the stigma of the plants in the Rutaceae family during the flowering period is effectively reduced, and the distant hybridization incompatibility between the Citrus grandis and the Clausena lansium is overcome. The reagent has little harm to the plants, has remarkable effect in overcoming the distant hybridization incompatibility, has wide application range, can be further used for overcoming the distant hybridization incompatibility of other plants in the Rutaceae family, and has important value for breeding and seed production of the plants in the Rutaceae family.

Description

Technical Field

[0001] This invention relates to the field of Rutaceae plant breeding technology, specifically to a reagent and its application method for overcoming incompatibility in distant hybridization of Rutaceae plants. Background Technology

[0002] Distant hybridization refers to hybridization between different species, genera, or more distantly related species in plant classification. Through distant hybridization, characteristics and traits of different species and genera can be combined, expanding genetic variation and creating new types of variations or even new species. However, for distantly related plants to hybridize, reproductive barriers before and after fertilization must be overcome. Prozygotic barriers refer to the complete inability of fertilization to occur after pollination of two fertile species, i.e., incompatibility exists. These barriers include two manifestations: pollen failing to germinate on the stigma or germinating abnormally, and pollen germinating normally but the pollen tube failing to grow properly in the style.

[0003] The genus *Citrus* in the Rutaceae family has undergone numerous intrageneric hybridizations during evolution, and most cultivated *Citrus* species are hybrids of two or more protozoan species. However, among Rutaceae species outside the *Citrus* genus, such as *Lampee* and *Murraya paniculata*, there are currently no reports of hybridization between them and other species, including those in the *Citrus* genus.

[0004] Citrus fruits, belonging to the Rutaceae family, are important fruit trees worldwide, holding a significant position in the national economy and possessing immense economic value. The citrus Huanglongbing (HLB) pathogen primarily infects Rutaceae plants, with cultivated Citrus, Citrus aurantium, and Citrus kumquat species serving as its hosts. Outside the Citrus genera, species like Wampee and Murraya paniculata exhibit strong resistance to HLB. Overcoming the barriers of distant hybridization and introducing HLB resistance into Citrus species would have significant research and economic value in helping citrus cultivation overcome the challenges posed by HLB.

[0005] Incompatibility between distant pollen and stigma is the first obstacle to distant hybridization, which has greatly limited the progress of distant hybridization breeding. Therefore, exploring efficient and widely applicable methods to overcome incompatibility in distant hybridization is an important part of distant hybridization breeding in the Rutaceae family. Summary of the Invention

[0006] In order to overcome the defects and deficiencies in the existing technology, the present invention provides a reagent and its application method for overcoming the incompatibility of distant hybridization in Rutaceae plants. The reagent used can overcome the incompatibility of distant hybridization, is simple and convenient to operate, and is easy to promote.

[0007] To achieve the objectives of this invention, EDTA or EGTA solution is used as the main component of the reagent. The specific technical solution is as follows:

[0008] A reagent for overcoming incompatibility in distant hybridization of Rutaceae plants, wherein the reagent is an EDTA or EGTA solution.

[0009] Furthermore, the reagent is a mixed solution of Tween-20 and EDTA, or a mixed solution of Tween-20 and EGTA.

[0010] Furthermore, in the mixed solution of Tween-20 and EDTA, the volume percentage of Tween-20 is 0.02%, and the concentration of EDTA is 1–10 mmol / L.

[0011] Furthermore, in the mixed solution of Tween-20 and EGTA, the volume percentage of Tween-20 was 0.02%, and the concentration of EGTA was 1–3 mmol / L.

[0012] Furthermore, the distant hybridization includes interspecific distant hybridization and intergeneric distant hybridization.

[0013] Another object of the present invention is to provide a method for overcoming incompatibility in distant hybridization of Rutaceae plants using the aforementioned reagent, the method comprising:

[0014] During the initial flowering period of Rutaceae plants, select unopened large buds, spray the aforementioned reagent onto their stigmas, and then proceed with pollination.

[0015] Furthermore, the reagent was sprayed twice onto the stigma of the unopened large buds.

[0016] Furthermore, the interval between the number of reagent drops is 30 minutes.

[0017] Furthermore, the present invention also provides the application of the reagent in overcoming incompatibility in distant hybridization of Rutaceae plants.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] This invention applies to distant hybridization incompatibility breeding in Rutaceae plants. In experiments breaking down distant hybridization incompatibility, EDTA and EGTA can interfere with the immune response between pollen and stigma by removing calcium ions. However, compared to plants in other families, calcium ions in Rutaceae plants not only participate in the immune response during hybridization incompatibility but also play an important role in multiple physiological processes such as pollen germination, pollen tube growth, and plant hormone regulation. Therefore, simply relying on EDTA and EGTA to remove calcium ions may interfere with the normal physiological processes of Rutaceae plants, thus negatively impacting the success of hybridization. In experiments with Rutaceae plants, excessive removal of calcium ions may lead to incomplete pollen germination or inhibited pollen tube growth, thereby affecting the hybridization success rate. Furthermore, the hybridization incompatibility mechanism in Rutaceae plants is more complex, involving more diverse physiological and molecular mechanisms than in other families, thus requiring more precise control of calcium ion concentration and timing of action in experiments.

[0020] This invention provides the application of EDTA or EGTA in overcoming the challenges of distant hybridization fertilization in Rutaceae plants. In this invention, EDTA or EGTA solution can effectively reduce the calcium content of the stigma in Citrus aurantium during flowering. 2+ This invention aims to improve the content of EDTA or EGTA solutions and overcome intergeneric hybridization incompatibility between Citrus aurantium and Wampee, promoting the germination, growth, and fertilization of Wampee pollen on the stigma of Citrus aurantium during flowering. Furthermore, it allows for precise control of the concentration and timing of EDTA or EGTA solutions to avoid negative impacts of calcium ion removal on other physiological processes, ensuring successful distant hybridization of Rutaceae plants and guaranteeing normal plant growth. Additionally, Tween-20 can be added to the reagent to increase the adhesion and permeability of the stigma to pollen. The method described in this invention is simple and convenient to operate, easy to promote, and has minimal damage to plants. It significantly overcomes distant hybridization incompatibility and has a wide range of applications, including Citrus aurantium and other Citrus species, as well as Wampee. Moreover, this method can be further used to overcome distant hybridization incompatibility in other Rutaceae plants, making it of significant value for Rutaceae breeding and seed production. Attached Figure Description

[0021] The disclosure of this invention will become more readily understood with reference to the accompanying drawings. It will be readily understood by those skilled in the art that these drawings are merely illustrative of the technical solutions of this invention and are not intended to limit the scope of protection of this invention.

[0022] Figure 1 To investigate the treatment of the stigmas of Citrus aurantiacus with EDTA solution, the results of aniline blue staining of the stigmas of Citrus aurantiacus were obtained 4 days after pollination with yellow-skinned pollen.

[0023] Figure 2 To investigate the use of EGTA solution to treat the stigmas of Citrus aurantiacus, the results of aniline blue staining of Citrus aurantiacus stigmas were obtained 4 days after pollination with yellow-skinned pollen.

[0024] Figure 3 The results of aniline blue staining of the stigmas of Citrus aurantiacus without any reagent treatment, 4 days after pollination with yellow-skinned pollen.

[0025] Figure 4 The results of aniline blue staining of the stigmas of Citrus aurantiacus were obtained 4 days after the stigmas were treated with 0.02% Tween-20 solution and pollinated with yellow-skinned flowers.

[0026] Figure 1-4 In this context, pt represents pollen tubes, and vb represents vascular bundles. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. All other embodiments obtained by those skilled in the art based on the embodiments in this application without inventive effort are within the scope of protection of this application.

[0028] The selective fertilization referred to in this invention, as described in *Modern Ecology*, is the phenomenon where a sperm nucleus with a specific genetic basis preferentially fertilizes an egg cell. Selective fertilization is mainly manifested in physiological, biochemical, and genetic characteristics, including incompatibility in distant hybridization and competition among multiple pollen sperm nuclei. In this invention, a distant hybridization between a four-season orange and a wampee is used as an example for illustration.

[0029] A reagent for overcoming incompatibility in distant hybridization of Rutaceae plants, wherein the reagent is an EDTA solution with a concentration of 1-10 mmol / L or an EGTA solution with a concentration of 1-3 mmol / L.

[0030] In the Rutaceae plant species described in this invention, the intergeneric hybridization incompatibility of Rutaceae plants is overcome, promoting germination, growth, entry into the stigma, and completion of the fertilization process. In this invention, the concentration of EDTA or EGTA solution used varies slightly for different Rutaceae plant species and can be adjusted according to actual conditions.

[0031] For example, the concentration of EDTA used for Citrus aurantiacus is 1-10 mmol / L, and the concentration of EGTA is 1-3 mmol / L. The method of application is to spray the reagent twice on the stigma of the Rutaceae plant during the bud stage in the morning, and then perform pollination after the second application.

[0032] When spraying, it is preferable to spray the reagent between 6:00 and 8:00 am on a sunny day, and the reagent should be added at 30-minute intervals.

[0033] The following detailed description, in conjunction with embodiments, illustrates the reagents and methods for overcoming incompatibility in distant hybridization of Rutaceae plants provided by this invention. However, these should not be construed as limiting the scope of protection of this invention.

[0034] Example 1

[0035] To overcome the incompatibility between citrus and wampee in distant hybridization, EDTA solution was used to treat citrus fruits. Specifically, the day before spraying the EDTA solution, the already opened flowers of the citrus fruits were removed and the fruits were then isolated by bagging. The following morning, before 8:00 AM, the unopened large buds were sprayed twice with the EDTA solution, which contained 0.02% Tween-20 and 1 mmol / L EDTA. The interval between the two sprays was 30 minutes.

[0036] After the second treatment, pollen was applied to the yellow-skinned flowers, and a pollination bag was used to prevent contamination from other pollen. Four days later, the pollination bag was removed, the style was picked and fixed with Carnoy's fixative, and the pollen germination was observed using aniline blue staining.

[0037] Example 2

[0038] The method is basically the same as in Example 1, except that the concentration of the EDTA solution is 3 mmol / L.

[0039] Example 3

[0040] The method is basically the same as in Example 1, except that the concentration of the EDTA solution is 5 mmol / L.

[0041] Example 4

[0042] The method is basically the same as in Example 1, except that the concentration of the EDTA solution is 7 mmol / L.

[0043] Example 5

[0044] The method is basically the same as in Example 1, except that the concentration of the EDTA solution is 10 mmol / L.

[0045] Example 6

[0046] To overcome the incompatibility between citrus and wampee in distant hybridization, EGTA solution was used to treat citrus fruits. Specifically, the day before spraying the EGTA solution, the already opened flowers of the citrus fruits were removed and the fruits were then isolated by bagging. The following morning at 8:00 AM, the unopened large buds were sprayed twice with the EGTA solution, which contained 0.02% Tween-20 by volume and 1 mmol / L EGTA. The interval between the two sprays was 30 minutes.

[0047] Example 7

[0048] It is basically the same as Example 6, except that the concentration of the EGTA solution is 3 mmol / L.

[0049] Figure 1 Four days after treating the stigmas of Citrus aurantiacus with EDTA solutions of different concentrations and pollinating with yellow-skinned flowers, the results of aniline blue staining of the stigmas were obtained.

[0050] Figure 2 Four days after treating the stigmas of Citrus aurantiacus with EGTA solutions of different concentrations and pollinating with yellow-skinned flowers, the results of aniline blue staining of the stigmas were obtained.

[0051] Comparative Example 1

[0052] The procedure is basically the same as in Example 1, except that the unopened flower buds of the four-season orange are not sprayed with the reagent.

[0053] Figure 3 The results of aniline blue staining of the stigmas of Citrus aurantiaca that were not treated with the reagent sprayed four days after pollination with yellow-skinned pollen.

[0054] Comparative Example 2

[0055] The treatment was basically the same as in Example 1, except that a 0.02% (v / v) Tween-20 solution was used as the reagent to spray the unopened buds of the four-season orange.

[0056] Figure 4 The results of aniline blue staining of the stigmas of Citrus aurantiacus were obtained 4 days after the stigmas were treated with 0.02% Tween-20 solution and pollinated with yellow-skinned flowers.

[0057] pass Figure 1-4 The comparison shows that after treating the stigma of the four-season orange with the EDTA or EGTA solution of the present invention, the pollen of the yellow peel can germinate, grow and enter the ovary to complete the fertilization process, which can overcome the incompatibility barrier between the four-season orange and the yellow peel in distant hybridization.

[0058] The above description, in conjunction with specific / preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various substitutions or modifications can be made to these described embodiments without departing from the inventive concept, and all such substitutions or modifications should be considered within the scope of protection of the present invention.

Claims

1. The application of a reagent in overcoming incompatibility in distant hybridization of Rutaceae plants, characterized in that, The reagent is a mixed solution of Tween-20 and EDTA, or a mixed solution of Tween-20 and EGTA; when the reagent is a mixed solution of Tween-20 and EDTA, the volume percentage of Tween-20 is 0.02% and the concentration of EDTA is 1~10 mmol / L; when the reagent is a mixed solution of Tween-20 and EGTA, the volume percentage of Tween-20 is 0.02% and the concentration of EGTA is 1~3 mmol / L.

2. The application of the reagent as described in claim 1 in overcoming incompatibility in distant hybridization of Rutaceae plants, characterized in that, The distant hybridization includes interspecific distant hybridization and intergeneric distant hybridization.

3. A method for using a reagent to overcome incompatibility in distant hybridization of Rutaceae plants, characterized in that, The process includes the following steps: at the initial flowering stage of Rutaceae plants, select unopened large buds, spray a reagent onto their stigmas, and then pollinate; the reagent is a mixed solution of Tween-20 and EDTA, or a mixed solution of Tween-20 and EGTA; when the reagent is a mixed solution of Tween-20 and EDTA, the volume percentage of Tween-20 is 0.02%, and the concentration of EDTA is 1~10 mmol / L; when the reagent is a mixed solution of Tween-20 and EGTA, the volume percentage of Tween-20 is 0.02%, and the concentration of EGTA is 1~3 mmol / L.

4. The method of using the reagent according to claim 3 to overcome incompatibility in distant hybridization of Rutaceae plants, characterized in that, The reagent was sprayed twice onto the stigma of the unopened large buds.

5. The method of using the reagent according to claim 4 to overcome incompatibility in distant hybridization of Rutaceae plants, characterized in that, The interval between the number of reagent sprayings is 30 minutes.

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