Reagent for overcoming distant hybridization incompatibility of rutaceous plants and use method thereof

Abstract

The invention discloses a reagent for overcoming distant hybridization incompatibility of rutaceous plants and a use method of the reagent. According to the method, the EDTA or EGTA solution is sprayed to the stigmas of the rutaceous plants, so that the content of Ca < 2 + > in the stigmas of the rutaceous plants in the flowering period can be effectively reduced, and the incompatibility of distant hybridization of the four-season oranges and the clausena lansium is overcome. The method is small in damage to plants, remarkable in effect of overcoming the distant hybridization incompatibility and wide in application range, can be further used for overcoming the distant hybridization incompatibility of other rutaceous plants, and has important value for breeding and seed production of the rutaceous plants.

Description

Technical Field

[0001] The invention relates to the technical field of Rutaceae plant breeding, and in particular to a reagent for overcoming the incompatibility of distant hybridization of Rutaceae plants and a use method thereof. Background Art

[0002] Distant hybridization refers to the hybridization between different species, genera or species with more distant genetic relationships in plant classification. Through distant hybridization, the characteristics and properties of different species and genera can be combined, genetic variation can be expanded, and new types of variation or even new species can be created. However, in order to hybridize between distantly related plants, reproductive barriers before and after fertilization must be overcome. Prozygotic barriers refer to the situation where the fertilization process of two fertile species cannot proceed after pollination, that is, there is an incompatibility phenomenon, including the inability of pollen to germinate on the stigma or abnormal germination, and the inability of pollen tubes to grow normally in the style although pollen can germinate normally.

[0003] The genus Citrus in the Rutaceae family has undergone many intra-genus hybridizations during its evolution, and most of the cultivated Citrus species are hybrids of two or more original species. However, among Rutaceae species outside the genus Citrus, such as Kumquat and Murraya paniculata, there are currently no reports of hybridization between them and other species, including Citrus species.

[0004] Citrus is an important fruit tree in the world, plays an important role in the national economy and has great economic value. Citrus Huanglongbing disease mainly infects plants of the Rutaceae family, and the cultivated plants of the genus Citrus, Trifoliate Orange and Fortunella are all its hosts. Outside the genus Citrus, Phellodendron and Osmanthus fragrans have extremely strong resistance to Huanglongbing disease. If the barrier of distant hybridization can be broken and Huanglongbing disease resistance can be introduced into citrus species, it will have great research value and economic value for the citrus planting industry to get rid of the trouble of Huanglongbing disease.

[0005] The incompatibility between distant pollen and stigma is the first obstacle to distant hybridization, which greatly limits the progress of distant hybridization breeding. Therefore, exploring efficient and widely applicable methods to overcome the incompatibility of distant hybridization is one of the important components of distant hybridization breeding in Rutaceae. Summary of the invention

[0006] In order to solve the defects and shortcomings in the above-mentioned prior art, the present invention provides a reagent for overcoming the incompatibility of distant hybridization of Rutaceae plants and a method for using the reagent. The reagent can overcome the incompatibility of distant hybridization, is simple and convenient to operate, and is easy to promote.

[0007] In order to achieve the purpose of the present invention, the present invention adopts EDTA or EGTA solution as the main component of the reagent, and its specific technical scheme is:

[0008] A reagent for overcoming the incompatibility of distant hybridization of Rutaceae plants, the reagent is 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 is 0.02%, and the concentration of EGTA is 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 the incompatibility of distant hybridization of Rutaceae plants using the reagent, the method comprising:

[0014] At the beginning of flowering of Rutaceae plants, select unopened large bud stage flowers, spray the agent on their stigmas and then perform pollination.

[0015] Furthermore, the reagent is sprayed on the stigma of the unopened large bud stage flowers twice.

[0016] Furthermore, the interval between the reagent dropwise additions is 30 minutes.

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

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention is applicable to the incompatibility breeding of distant hybridization of Rutaceae plants. In the experiment of breaking the incompatibility of distant hybridization, EDTA and EGTA can intervene the immune response between pollen and stigma by removing calcium ions. However, compared with plants of other families, in Rutaceae plants, calcium ions 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 process of Rutaceae plants, thereby having a negative impact on the success of hybridization. In the experiment of Rutaceae plants, excessive removal of calcium ions may cause incomplete pollen germination or obstructed pollen tube growth, thereby affecting the hybridization success rate. In addition, the hybridization incompatibility mechanism of Rutaceae plants is relatively complex, and the physiological and molecular mechanisms involved are more diverse than those of plants of other families, so it is necessary to more finely control the concentration and timing of calcium ions in the experiment.

[0020] The present invention provides the use of EDTA or EGTA in overcoming the fertilization of distant hybridization of Rutaceae plants. In the present invention, on the one hand, the use of EDTA or EGTA solution can effectively reduce the stigma Ca in the flowering period of four-season orange. 2+ content, and overcome the inter-genus hybridization incompatibility of four-season orange and yellow peel, promote yellow peel pollen to germinate, grow, enter the stigma and complete the fertilization process on the stigma of four-season orange during flowering period; on the other hand, the concentration and use time of EDTA or EGTA solution are precisely regulated to avoid the negative impact of calcium ion removal on other physiological processes, so that the distant hybridization of Rutaceae plants can be smoothly carried out and the normal growth of plants can be ensured. At the same time, Tween-20 can also be added to the reagent to increase the adhesion and permeability of the stigma to pollen. The method of the present invention is simple and convenient to operate, easy to promote, has little harm to plants, has a significant effect of overcoming the incompatibility of distant hybridization, has a wide range of applications, and can be applied to citrus and yellow peel plants such as four-season orange; and the method can be further used to overcome the incompatibility of distant hybridization of other plants of Rutaceae, which is of great value to the breeding and seed production of Rutaceae plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The disclosure of the present invention will become more easily understood with reference to the accompanying drawings. It is easy for those skilled in the art to understand that these drawings are only used to illustrate the technical solutions of the present invention and are not intended to limit the protection scope of the present invention.

[0022] Figure 1 In order to use EDTA solution to treat the stigma of Four Seasons Orange, the stigma of Four Seasons Orange was stained with aniline blue 4 days after being pollinated with yellow pollen.

[0023] Figure 2 In order to use EGTA solution to treat the stigma of Four Seasons Orange, the stigma of Four Seasons Orange was dyed with aniline blue 4 days after being pollinated with yellow pollen.

[0024] Figure 3 This is the stigma of the Four Seasons Mandarin Orange that has not been treated with any reagents. The result of aniline blue staining of the Four Seasons Mandarin Orange stigma was 4 days after being pollinated with yellow pollen.

[0025] Figure 4 The stigma of Four Seasons Mandarin Orange was treated with 0.02% Tween-20 solution by volume, and the stigma of Four Seasons Mandarin Orange was stained with aniline blue 4 days after being pollinated with yellow pollen.

[0026] Figure 1-4 In the figure, pt is the pollen tube and vb is the vascular bundle. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection of this application.

[0028] The selective fertilization referred to in the present invention is recorded in Modern Ecology as the phenomenon that sperm nuclei with a specific genetic basis preferentially fertilize egg cells. Selective fertilization is mainly manifested in physiological, biochemical and genetic characteristics, including distant hybridization incompatibility, competition between multiple pollen sperm nuclei, etc. In the embodiment of the present invention, the distant hybridization of Siji Mandarin Orange and Wampee is used as an example for explanation.

[0029] A reagent for overcoming the incompatibility of distant hybridization of Rutaceae plants. 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 the present invention, the intergeneric hybridization incompatibility of the Rutaceae plants is overcome to promote germination, growth, entry into the stigma and completion of the fertilization process. In the present invention, for different species of Rutaceae plants, the concentration of the EDTA or EGTA solution used is slightly different and can be adjusted according to actual conditions.

[0031] For example, the concentration of EDTA used for four-season orange is 1-10mmol / L, and the concentration of EGTA is 1-3mmol / L. The method of using the reagent is to spray the reagent twice on the stigma of the bud stage of the Rutaceae plant in the morning, and pollinate after the second drip.

[0032] When spraying, it is preferred to spray the reagent between 6:00 and 8:00 in the morning on a sunny day, and the dripping interval of the reagent is 30 minutes.

[0033] The reagents and methods of use for overcoming the incompatibility of distant hybridization of Rutaceae plants provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1

[0035] The EDTA solution was used to treat the four-season orange to overcome the incompatibility of the four-season orange and the yellow peel distant hybridization. Specifically, the day before spraying the EDTA solution, the flowers of the four-season orange that have already opened were first removed and then bagged for isolation. Before 8:00 am the next day, the unopened large bud stage flowers were sprayed with EDTA solution twice. The EDTA solution contained 0.02% Tween-20 by volume and 1 mmol / L EDTA. The treatment interval between the two sprayings was 30 minutes.

[0036] After the second treatment, the yellow peel was pollinated and covered with pollination bags to prevent contamination by other pollens. Four days later, the pollination bags were removed, the styles were picked and fixed with Carnoy's fixative, and the pollen germination was observed by aniline blue staining.

[0037] Example 2

[0038] It is basically the same as 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 Example 1, except that the concentration of the EDTA solution is 5 mmol / L.

[0041] Example 4

[0042] It is basically the same as 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 Example 1, except that the concentration of the EDTA solution is 10 mmol / L.

[0045] Example 6

[0046] EGTA solution was used to treat the four-season orange to overcome the incompatibility of the four-season orange and the yellow peel distant hybridization. Specifically, the day before spraying the EGTA solution, the four-season orange flowers that had already opened were first removed and then bagged for isolation. At 8:00 am the next day, the unopened large bud stage flowers were sprayed with EGTA solution twice. The EGTA solution contained 0.02% Tween-20 by volume and 1 mmol / L EGTA. The treatment interval between the two sprayings 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 The stigmas of Four Seasons Orange were treated with EDTA solutions of different concentrations and then stained with aniline blue 4 days after being pollinated with yellow pollen.

[0050] Figure 2 The stigmas of Mandarin orange were treated with EGTA solutions of different concentrations and then stained with aniline blue 4 days after being pollinated with yellow pollen.

[0051] Comparative Example 1

[0052] The method is basically the same as Example 1, except that the unopened flower buds of the four-season orange are not treated by spraying the reagent.

[0053] Figure 3 This is the result of aniline blue staining of the stigma of the Four Seasons Mandarin Orange that was not sprayed with reagents and was 4 days after being pollinated with yellow pollen.

[0054] Comparative Example 2

[0055] The method is basically the same as Example 1, except that a 0.02% by volume Tween-20 solution is used as a reagent to spray the unopened flower buds of the four-season orange.

[0056] Figure 4 The stigma of Four Seasons Mandarin Orange was treated with 0.02% Tween-20 solution by volume, and the stigma of Four Seasons Mandarin Orange was stained with aniline blue 4 days after being pollinated with yellow pollen.

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

[0058] The above contents are further detailed descriptions of the present invention in combination with specific / preferred implementations, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, they can also make several substitutions or modifications to these described embodiments without departing from the concept of the present invention, and these substitutions or modifications should be regarded as belonging to the protection scope of the present invention.

Claims

1. A reagent for overcoming the incompatibility of distant hybridization of Rutaceae plants, characterized in that: The reagent is EDTA or EGTA solution.

2. The reagent for overcoming the incompatibility of distant hybridization of Rutaceae plants according to claim 1, characterized in that: The reagent is a mixed solution of Tween-20 and EDTA, or a mixed solution of Tween-20 and EGTA.

3. The reagent for overcoming the incompatibility of distant hybridization of Rutaceae plants according to claim 2, characterized in that: 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.

4. The reagent for overcoming the incompatibility of distant hybridization of Rutaceae plants according to claim 2, characterized in that: 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.

5. A reagent for overcoming the incompatibility of distant hybridization of Rutaceae plants according to claims 1-4, characterized in that: The distant hybridization includes interspecific distant hybridization and intergeneric distant hybridization.

6. A method for using a reagent for overcoming incompatibility of distant hybridization of Rutaceae plants as claimed in any one of claims 1 to 5, characterized in that: The method comprises the following steps: selecting unopened large bud stage flowers of Rutaceae plants at the beginning of flowering, spraying the reagent on the stigma of the flowers and then pollinating the flowers.

7. The method for using a reagent for overcoming incompatibility of distant hybridization of Rutaceae plants according to claim 6, characterized in that: The agent was sprayed twice on the stigma of the unopened large bud stage flowers.

8. The method for using a reagent for overcoming incompatibility of distant hybridization of Rutaceae plants according to claim 7, characterized in that: The interval between the spraying times of the reagent is 30 minutes.

9. Use of a reagent for overcoming incompatibility in distant hybridization of Rutaceae plants as claimed in any one of claims 1 to 5 in distant hybridization of Rutaceae plants.

10. Use of the method for overcoming the incompatibility of Rutaceae plants in distant hybridization as claimed in any one of claims 6 to 8 in distant hybridization of Rutaceae plants.

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