High-yield rice breeding method with saline-alkaline tolerance gene introduction

Abstract

The invention discloses a saline-alkaline tolerance gene introduced rice high-yield breeding method. The method comprises the following steps: S1, gene screening and cloning: screening related genes with saline-alkaline tolerance; s2, vector construction: inserting the screened saline-alkaline tolerant gene into a proper expression vector; s3, transforming rice cells: introducing the vector carrying the saline-alkaline tolerant gene into the rice cells; s4, screening positive plants: confirming that the rice cells with the saline-alkaline tolerant genes are successfully transferred, and selecting the positive plants from the rice cells for next-step culture; s5, tissue culture and development: placing the positive plants on a sterile culture medium for tissue culture, and enabling the positive plants to develop into complete plants; s6, planting and harvesting: transplanting and planting the obtained transgenic rice plants, and harvesting the transgenic rice plants to obtain rice seeds. The saline-alkali soil-resistant gene disclosed by the invention is stronger in saline-alkali resistance, the yield is not influenced after the gene is planted in saline-alkali soil, and high-yield rice can be obtained.

Description

Technical Field

[0001] The invention relates to the technical field of rice breeding, in particular to a high-yield rice breeding method with the introduction of salt-alkali tolerance genes. Background Art

[0002] Rice is sensitive to salt and alkali, and soil salinization is one of the important reasons that affect rice growth. A large number of scientific studies have shown that the development of saline-alkali soil for rice planting is an effective measure for successful soil management, improvement and utilization. Salt-alkali stress has a great inhibitory effect on the growth and development of rice. Salt harms crops in the form of osmotic and ion stress, seriously affecting the metabolism of crops, inhibiting the growth and development of crops, and ultimately leading to plant death, making the yield and quality of rice in saline-alkali land lower than that of non-saline-alkali land. Salt-alkali stress has different effects on different stages of rice growth. During the reproductive growth stage of rice, salt-alkali stress seriously affects the normal differentiation of rice panicles and the formation of spikelets, resulting in a decrease in grain yield and a decrease in rice quality.

[0003] The invention discloses a rapid screening method for salt-alkali tolerant rice breeding materials, which is disclosed in the publication number CN114731799A and relates to the field of salt-alkali tolerant rice breeding. The technical scheme comprises the following steps: Step 1: Raw material screening: Select rice seeds with uniform size, round outer surface, no damage and no mildew. Ten varieties of seeds are selected, 10 seeds of each variety, and a total of 200 seeds are selected; Step 2: Raw material pretreatment: Rinse the selected rice seeds with an ethanol solution, and then rinse them again with pure water after rinsing; Step 3: Group experiment: The treated rice seeds are grouped and placed in a culture dish for breeding, and data a, data b and data c are summarized and compared to establish a salt-alkali tolerant rice screening model, and the data detected by rice starch is used as a judgment standard, so as to correct the established salt-alkali tolerant rice screening model, so as to greatly improve the subsequent salt-alkali tolerant screening efficiency of rice seeds.

[0004] The existing technology can only achieve the goal of planting ordinary rice seeds in saline-alkali land and then screening the ordinary seeds with higher yields. It cannot obtain rice seeds that are tolerant to saline-alkali land and have higher yields. Therefore, we propose a high-yield rice breeding method with the introduction of salt-alkali tolerance genes. Summary of the invention

[0005] The purpose of the present invention is to provide a high-yield rice breeding method with introduced salt-alkali tolerance gene to solve the problems in the prior art.

[0006] To achieve the above object, the present invention provides the following technical solution: a method for high-yield rice breeding with salt-alkali tolerance gene introduction, comprising the following steps:

[0007] S1. Gene screening and cloning: Screening of genes related to salt-alkali tolerance;

[0008] S2. Vector construction: insert the selected salt-alkali tolerance gene into a suitable expression vector;

[0009] S3, transforming rice cells: introducing the vector carrying the salt-alkali tolerance gene into rice cells;

[0010] S4, screening positive plants: confirming the rice cells that have been successfully transferred with the salt-alkali tolerance gene, and selecting positive plants for the next step of cultivation;

[0011] S5, tissue culture and development: Place the positive plants on sterile culture medium for tissue culture to develop them into complete plants;

[0012] S6, planting and harvesting: transplanting the obtained transgenic rice plants and harvesting them to obtain rice seeds;

[0013] S7. Continuous self-pollination: Use continuous self-pollination for multiple generations to obtain rice with stable inherited salt-alkali tolerance genes.

[0014] Preferably, the salt-alkali tolerance gene in S1 is obtained from existing plants or artificially synthesized.

[0015] Preferably, the vector in S2 includes a plasmid or a viral vector.

[0016] Preferably, the method for introducing the vector in S3 is Agrobacterium-mediated method or pollen tube channel method.

[0017] Preferably, the Agrobacterium-mediated method comprises the following steps:

[0018] S31, introducing the constructed Ti plasmid into Agrobacterium;

[0019] S32, selecting rice material;

[0020] S33, co-culturing the Agrobacterium containing the recombinant Ti plasmid with the prepared rice material for a period of time, during which the Agrobacterium will infect the rice cells and transfer the T-DNA region to the rice chromosome;

[0021] S34. Antibiotic screening: Add antibiotics to the culture medium so that only cells that have successfully integrated the Ti plasmid can survive.

[0022] Preferably, the screening of positive plants in S4 is performed by using PCR detection and Southern hybridization to confirm the rice cells into which the salt-alkali tolerance gene has been successfully transferred.

[0023] Preferably, the continuous selfing of S7 comprises the following steps:

[0024] S71. Selecting a female parent: selecting a healthy rice plant as a female parent;

[0025] S72. Remove stamens: About two days before flowering, gently shake the rice ears with your hands to let the anthers fall off;

[0026] S73, wrapping: wrapping the rice ears with the stamens removed with plastic bags;

[0027] S74. Wait until the flowers on the rice ears are fully open before opening the packaging;

[0028] S75, Artificial pollination: Dip the mature pollen from the same plant and apply it to the stigma of the already opened pistil;

[0029] S76. Harvesting seeds: After maturity, harvest the seeds.

[0030] Preferably, the method further includes S8, testing: testing food safety and ecological safety to ensure that the genetically modified rice is harmless to human health and the ecological environment.

[0031] Compared with the prior art, the invention has the following beneficial effects: screening relevant genes with salt-alkali resistance; inserting the screened salt-alkali resistance genes into a suitable expression vector; introducing the vector carrying the salt-alkali resistance gene into rice cells; confirming the rice cells into which the salt-alkali resistance gene has been successfully transferred, and selecting positive plants therefrom for further cultivation; placing the positive plants on a sterile culture medium for tissue culture to develop them into complete plants; transplanting and planting the obtained transgenic rice plants, and harvesting them to obtain rice seeds; adopting a continuous multi-generation self-pollination method to obtain rice with stably inherited salt-alkali resistance genes; the rice seeds obtained in the present application have stronger salt-alkali resistance because they have salt-alkali resistance genes, and the yield will not be affected after being planted in saline-alkali land, so that high-yield rice can be obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0033] Figure 1 It is a flow chart of the present invention. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention.

[0035] See also Figure 1 In an embodiment of the present invention, a method for high-yield rice breeding with introduction of salt-alkali tolerance gene comprises the following steps:

[0036] S1. Gene screening and cloning: Screening of genes related to salt-alkali tolerance;

[0037] S2. Vector construction: insert the selected salt-alkali tolerance gene into a suitable expression vector;

[0038] S3, transforming rice cells: introducing the vector carrying the salt-alkali tolerance gene into rice cells;

[0039] S4, screening positive plants: confirming the rice cells that have been successfully transferred with the salt-alkali tolerance gene, and selecting positive plants for the next step of cultivation;

[0040] S5, tissue culture and development: Place the positive plants on sterile culture medium for tissue culture to develop them into complete plants;

[0041] S6, planting and harvesting: transplanting the obtained transgenic rice plants and harvesting them to obtain rice seeds;

[0042] S7. Continuous self-pollination: Use continuous self-pollination for multiple generations to obtain rice with stably inherited salt-alkali tolerance genes; conduct genetic stability tests on the offspring of continuous self-pollination for multiple generations to ensure that the salt-alkali tolerance characteristics can be stably inherited.

[0043] Preferably, the salt-alkali tolerance gene in S1 is obtained from existing plants or artificially synthesized.

[0044] Preferably, the vector in S2 includes a plasmid or a viral vector.

[0045] Preferably, the method for introducing the vector in S3 is Agrobacterium-mediated method or pollen tube channel method.

[0046] Preferably, the Agrobacterium-mediated method comprises the following steps:

[0047] S31, introducing the constructed Ti plasmid into Agrobacterium;

[0048] S32, selecting rice material;

[0049] S33, co-culturing the Agrobacterium containing the recombinant Ti plasmid with the prepared rice material for a period of time, during which the Agrobacterium will infect the rice cells and transfer the T-DNA region to the rice chromosome;

[0050] S34. Antibiotic screening: Add antibiotics to the culture medium so that only cells that have successfully integrated the Ti plasmid can survive.

[0051] Preferably, the screening of positive plants in S4 is performed by using PCR detection and Southern hybridization to confirm the rice cells into which the salt-alkali tolerance gene has been successfully transferred.

[0052] Preferably, the continuous selfing of S7 comprises the following steps:

[0053] S71. Selecting a female parent: selecting a healthy rice plant as a female parent;

[0054] S72. Remove stamens: About two days before flowering, gently shake the rice ears with your hands to let the anthers fall off;

[0055] S73, wrapping: wrapping the rice ears with the stamens removed with plastic bags;

[0056] S74. Wait until the flowers on the rice ears are fully open before opening the packaging;

[0057] S75, Artificial pollination: Dip the mature pollen from the same plant and apply it to the stigma of the already opened pistil;

[0058] S76. Harvesting seeds: After maturity, harvest the seeds.

[0059] Preferably, the method further includes S8, testing: testing food safety and ecological safety to ensure that the genetically modified rice is harmless to human health and the ecological environment.

[0060] The working principle of the present invention is as follows: screening relevant genes with salt-alkali resistance; inserting the screened salt-alkali resistance genes into a suitable expression vector; introducing the vector carrying the salt-alkali resistance gene into rice cells; confirming the rice cells into which the salt-alkali resistance gene has been successfully transferred, and selecting positive plants therefrom for further cultivation; placing the positive plants on a sterile culture medium for tissue culture to develop them into complete plants; transplanting and planting the obtained transgenic rice plants, and harvesting them to obtain rice seeds; adopting a continuous multi-generation self-pollination method to obtain rice with stably inherited salt-alkali resistance genes; the rice seeds obtained in the present application have stronger salt-alkali resistance performance because they have salt-alkali resistance genes, and the yield will not be affected after being planted in saline-alkali land, so that high-yield rice can be obtained.

[0061] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for high-yield rice breeding with introduction of salt-alkali tolerance gene, characterized in that: The following steps are involved: S1. Gene screening and cloning: Screening of genes related to salt-alkali tolerance; S2. Vector construction: insert the selected salt-alkali tolerance gene into a suitable expression vector; S3, transforming rice cells: introducing the vector carrying the salt-alkali tolerance gene into rice cells; S4, screening positive plants: confirming the rice cells that have been successfully transferred with the salt-alkali tolerance gene, and selecting positive plants for the next step of cultivation; S5, tissue culture and development: Place the positive plants on sterile culture medium for tissue culture to develop them into complete plants; S6, planting and harvesting: transplanting the obtained transgenic rice plants and harvesting them to obtain rice seeds; S7. Continuous self-pollination: Use continuous self-pollination for multiple generations to obtain rice with stable inherited salt-alkali tolerance genes.

2. The method for high-yield rice breeding with salt-alkali tolerance gene introduction according to claim 1, characterized in that: The salt-alkali tolerance gene in S1 is obtained from existing plants or artificially synthesized.

3. The high-yield rice breeding method with salt-alkali tolerance gene introduction according to claim 1, characterized in that: The vector in S2 includes plasmid and viral vector.

4. The high-yield rice breeding method with salt-alkali tolerance gene introduction according to claim 1, characterized in that: The method for introducing the vector in S3 is Agrobacterium-mediated method and pollen tube pathway method.

5. The high-yield rice breeding method with salt-alkali tolerance gene introduction according to claim 4, characterized in that: The Agrobacterium-mediated method comprises the following steps: S31, introducing the constructed Ti plasmid into Agrobacterium; S32, selecting rice material; S33, co-culturing the Agrobacterium containing the recombinant Ti plasmid with the prepared rice material for a period of time, during which the Agrobacterium will infect the rice cells and transfer the T-DNA region to the rice chromosome; S34. Antibiotic screening: Add antibiotics to the culture medium so that only cells that have successfully integrated the Ti plasmid can survive.

6. The high-yield rice breeding method with salt-alkali tolerance gene introduction according to claim 1, characterized in that: The screening of positive plants in S4 is carried out by using PCR detection and Southern hybridization to confirm the rice cells into which the salt-alkali tolerance gene has been successfully transferred.

7. The high-yield rice breeding method with salt-alkali tolerance gene introduction according to claim 1, characterized in that: The continuous selfing of S7 comprises the following steps: S71. Selecting a female parent: selecting a healthy rice plant as a female parent; S72. Remove stamens: About two days before flowering, gently shake the rice ears with your hands to let the anthers fall off; S73, wrapping: wrapping the rice ears with the stamens removed with plastic bags; S74. Wait until the flowers on the rice ears are fully open before opening the packaging; S75, Artificial pollination: Dip the mature pollen from the same plant and apply it to the stigma of the already opened pistil; S76. Harvesting seeds: After maturity, harvest the seeds.

8. The high-yield rice breeding method with salt-alkali tolerance gene introduction according to claim 1, characterized in that: It also includes S8, testing: testing food safety and ecological safety to ensure that genetically modified rice is harmless to human health and the ecological environment.

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