Method for inducing soybean polyploidy by colchicine

Abstract

The invention relates to the field of soybean breeding, and particularly discloses a method for inducing soybean polyploidy by colchicine, which comprises the following steps: soaking soybean seeds in a colchicine solution, then taking out and cleaning, then placing the seeds in special water storage paper, and transplanting the seeds into a seedling raising pot after soybean germination. According to the breeding method, autotetraploid soybeans can be rapidly created on a large scale, and the survival rate of soybean seeds soaked in colchicine can be effectively increased.

Description

Technical Field

[0001] The invention relates to the field of soybean polyploid breeding, and in particular to a method for inducing soybean polyploidy by using colchicine. Background Art

[0002] Soybean is an annual diploid and one of the major food crops and cash crops in the world. Its output in 2023 was about 375.4 million tons. It is of great significance in food production and feed processing.

[0003] The main difficulties currently faced by soybeans include insufficient arable land and insufficient germplasm resources, which have greatly restricted the breeding of soybean varieties, especially high-quality soybeans. At the same time, due to the lack of research resources related to high-quality soybean germplasm, the development of high-quality soybeans is still relatively slow. Polyploid crops have the advantages of rich nutritional value and strong stress resistance. The hybrid advantages brought by genome doubling can also expand the germplasm, which is an important method for plant germplasm innovation and new variety breeding. Among them, colchicine is the most widely used chemical mutagen, which can inhibit mitosis, destroy spindles, cause chromosomes to stagnate in metaphase, and double chromosomes. In recent years, colchicine-induced polyploidy has been widely used in various economic crops and food crops, but the survival rate of seeds will be greatly reduced after soaking in colchicine. At present, there is no relevant technology for large-scale induction of polyploid soybeans using colchicine.

[0004] Therefore, it is necessary to design a new method of inducing soybean polyploidy using colchicine to solve the problem of low survival rate of soybean after soaking in colchicine and fill the gap in large-scale induction of soybean polyploidy. Summary of the invention

[0005] In view of this, the present invention proposes a method for inducing soybean polyploidy by using colchicine, so as to solve the problem of low survival rate of soybean after soaking in colchicine and fill the gap of large-scale induction of soybean polyploidy.

[0006] On the one hand, the present invention provides a method for inducing soybean polyploidy using colchicine, comprising:

[0007] Selecting soybean seeds, placing the soybean seeds in a colchicine solution for soaking, and obtaining soaked soybean seeds;

[0008] The soaked soybean seeds are post-processed, and then the soybean seeds are wrapped with water storage paper to obtain seed bags;

[0009] The seed package is placed in a room temperature environment, and after the seeds germinate, the germinated seeds are transplanted into a seedling pot, and after true leaves are grown, surviving individual plants are identified and screened to obtain soybean polyploid seedlings.

[0010] Furthermore, it is characterized in that the mass concentration of the colchicine solution is 0.05%-0.2%.

[0011] Furthermore, the soaking time is 6-12 hours, and the soaking environment is a dark environment without light.

[0012] Furthermore, the post-treatment is specifically to wrap the soaked soybean seeds in a mesh bag and rinse them with running water for 1 hour.

[0013] Furthermore, the water storage paper is formed by stacking newspapers and toilet paper, and the seed bag wrapping method is specifically as follows: stacking a layer of water storage paper on the water storage paper, arranging the soybean seeds neatly and then rolling up.

[0014] Furthermore, the specific method of identification and screening is:

[0015] Take leaves from the surviving individual plants and place them in culture dishes, add 250 μL of lysis solution to the culture dishes, and chop the leaves into a paste with a blade;

[0016] 250 μL of lysis solution was added to the culture dish again, followed by rinsing with ultrapure water, and filtered through a 50-mesh cell sieve into a flow tube in a flow cytometer for detection, and soybean polyploid seedlings were screened out based on the results.

[0017] Furthermore, the soil in the seedling pot is made of loess and nutrient soil in a volume ratio of 1:1.

[0018] On the other hand, the present invention provides a method for inducing soybean polyploidy using colchicine and its application in soybean polyploidy breeding.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. The plant material of homologous tetraploid soybean was successfully created by the method of soaking in colchicine, enriching the gene pool of soybean and providing a basis for breeding high-yield, high-quality and strong stress-resistant soybean seeds.

[0021] 2. The seedling raising method of wrapping the seed bag with water-retaining paper effectively improves the survival rate of soybean seeds after being soaked in colchicine, so that the present invention can quickly and large-scale create autotetraploid soybeans. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. Moreover, the same reference symbols are used throughout the accompanying drawings to represent the same components. In the accompanying drawings:

[0023] Figure 1 A flow chart of a method for inducing soybean polyploidy using colchicine provided in an embodiment of the present invention.

[0024] Figure 2 This is a comparison diagram of plant morphology after subsequent cultivation of the self-pollinated seeds of the surviving diploid soybean and the self-pollinated seeds of the tetraploid soybean in the examples of the present invention.

[0025] Figure 3 This is a comparison diagram of soybean leaves after subsequent cultivation of the self-pollinated seeds of the surviving diploid soybean and the self-pollinated seeds of the tetraploid soybean in the examples of the present invention.

[0026] Figure 4 This is a comparison of soybean petal sizes after subsequent cultivation of surviving self-pollinated diploid soybean seeds and self-pollinated tetraploid soybean seeds in an embodiment of the present invention.

[0027] Figure 5 This is a comparison diagram of soybean seed sizes after subsequent cultivation of self-pollinated seeds of surviving diploid soybeans and self-pollinated seeds of tetraploid soybeans in an embodiment of the present invention.

[0028] Figure 6 It is a comparison chart of soybean seed quality traits after subsequent cultivation of self-pollinated seeds of surviving diploid soybeans and self-pollinated seeds of tetraploid soybeans in an embodiment of the present invention.

[0029] Figure 7 A step diagram of a seed packet germination and seedling cultivation method provided in an embodiment of the present invention.

[0030] Figure 8 This is a peak diagram of the genome of soybean plants with different ploidy detected by the flow cytometer provided in the embodiment of the present invention. DETAILED DESCRIPTION

[0031] Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art. It should be noted that, in the absence of conflict, the embodiments of the present invention and the features described in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0032] Soybean is an annual diploid and one of the main food crops and cash crops in the world. Its output in 2023 is about 375.4 million tons. It is of great significance in the fields of food production and feed processing. The main difficulties currently faced by soybeans include insufficient arable land and insufficient germplasm resources, which have greatly restricted the breeding of soybean varieties, especially high-quality soybeans. At the same time, due to the lack of research resources for high-quality soybean germplasm, the development of high-quality soybeans is still relatively slow. Polyploid crops have the advantages of rich nutritional value and strong stress resistance. The hybrid advantages brought by genome doubling can also expand the germplasm, which is an important method for plant germplasm innovation and new variety breeding. Among them, colchicine is the most widely used chemical mutagen, which can inhibit mitosis, destroy spindles, cause chromosomes to stagnate in metaphase, and double chromosomes. In recent years, colchicine-induced polyploidy has been widely used in various cash crops and food crops, but the survival rate of seeds will be greatly reduced after soaking in colchicine. At present, there is no relevant technology for inducing polyploid soybeans on a large scale using colchicine. Therefore, it is necessary to design a new method of inducing soybean polyploidy using colchicine to solve the problem of low survival rate of soybean after soaking in colchicine and fill the gap in large-scale induction of soybean polyploidy.

[0033] On the one hand, in some embodiments of the present invention, a method for inducing soybean polyploidy using colchicine is characterized by comprising the following steps:

[0034] Selecting soybean seeds, placing the soybean seeds in a colchicine solution for soaking, and obtaining soaked soybean seeds;

[0035] The soaked soybean seeds are post-processed, and then the soybean seeds are wrapped with water storage paper to obtain seed bags;

[0036] The seed package is placed in a room temperature environment, and after the seeds germinate, the germinated seeds are transplanted into a seedling pot, and after true leaves are grown, surviving individual plants are identified and screened to obtain soybean polyploid seedlings.

[0037] Specifically, when selecting seeds, choose soybean seeds with full grains and strong vitality.

[0038] It is understandable that when soybean seeds or seedlings are soaked in colchicine, after entering the cells, colchicine binds to tubulin, which is the basic unit of spindle microtubules, thereby inhibiting the formation of the spindle. Since the spindle cannot be formed normally, in the late stage of mitosis, although the chromosomes can replicate normally (DNA replication), because there is no traction of the spindle, the chromosomes cannot move to the two poles of the cell, resulting in the doubling of the number of chromosomes in the cell, thereby obtaining homologous tetraploid soybeans.

[0039] It is understandable that compared with diploid soybeans, autotetraploid soybeans have doubled chromosome sets and increased gene dosage, which means that the number of copies of many genes related to growth and development, yield formation, quality and stress resistance has increased, thereby increasing the expression of these genes and enhancing the corresponding physiological functions. For example, the increase in the dosage of some key genes involved in photosynthesis can increase the chlorophyll content in the leaves, enhance the efficiency of photosynthesis, synthesize more organic matter, provide a more sufficient material basis for plant growth and seed development, and ultimately increase yield; and after the chromosomes double, the cell volume increases, which in turn leads to the enlargement of organs such as leaves, stems, and seeds. The increase in leaf area can improve the efficiency of photosynthesis and product accumulation; the increase in seed volume and weight allows the seeds to store more nutrients, which is beneficial to the growth and development of seedlings and improves the stress resistance of seedlings.

[0040] In some embodiments of the present invention, the mass concentration of the colchicine solution is 0.05%-0.2%, and the mass concentration of the colchicine solution is preferably 0.2%.

[0041] In some embodiments of the present invention, the soaking time is 6-12 hours, and the soaking environment is a dark environment without light; the soaking time is preferably 12 hours.

[0042] In some embodiments of the present invention, the post-treatment is specifically to wrap the soaked soybean seeds in a mesh bag and rinse them with running water for 1 hour.

[0043] It is understandable that colchicine is a toxic alkaloid. The soaked soybean seeds were wrapped in a mesh bag and rinsed with running water for 1 hour. The main purpose was to remove as much colchicine as possible from the surface and inside of the seeds. If too much colchicine remains on the seeds, it may continue to toxicize seed cells during subsequent germination and growth, affecting the normal germination of seeds and the growth and development of seedlings.

[0044] It is understandable that using a mesh bag to wrap the seeds for flushing can ensure that the seeds have good air permeability during the flushing process. The seeds still need to respire aerobically after being soaked in colchicine. If there is not enough oxygen supply, the seeds may be damaged due to lack of oxygen. At the same time, the mesh bag allows the water flow to fully contact each seed, ensuring the uniformity of flushing and avoiding differences in seed germination and growth due to excessive local colchicine residue.

[0045] like Figure 7 As shown, in some embodiments of the present invention, the water storage paper is formed by stacking newspapers and toilet paper, and the seed bag wrapping method is specifically: stacking a layer of water storage paper on the water storage paper, arranging the soybean seeds neatly and then rolling them up.

[0046] It is understandable that newspapers and toilet paper both have certain water absorption and moisture retention properties. In the process of seed germination, suitable humidity is one of the key conditions. This seed bag can provide a relatively stable moist environment for seeds, preventing the seeds from losing water and shrinking due to rapid evaporation of water, helping to maintain the vitality of the seeds and promote smooth germination of seeds.

[0047] It is understandable that newspapers and toilet paper are not completely sealed materials. They have certain pores, which can ensure that the seeds can exchange gases inside the bag. Seed germination requires oxygen for respiration. Such seed bags can allow enough oxygen to enter while expelling carbon dioxide to maintain normal respiratory metabolism of the seeds.

[0048] It is understandable that the stacked newspapers and toilet paper can play a certain role in temperature buffering. When the external environment temperature fluctuates, the temperature change in the seed bag is relatively mild, reducing the adverse effect of temperature on seed germination and providing a relatively mild microenvironment for seed germination.

[0049] It is understandable that seed packets can isolate seeds from possible external pollutants, pests, etc. to ensure smooth germination of seeds.

[0050] In some embodiments of the present invention, the specific method of identification and screening is:

[0051] Take leaves from the surviving individual plants and place them in culture dishes, add 250 μL of lysis solution to the culture dishes, and chop the leaves into a paste with a blade;

[0052] 250 μL of lysis solution was added to the culture dish again, followed by rinsing with ultrapure water, and filtered through a 50-mesh cell sieve into a flow tube in a flow cytometer for detection, and soybean polyploid seedlings were screened out based on the results.

[0053] The lysis solution is a commercially available lysis solution of model CyStain UV Ploidy DAPI.

[0054] Specifically, if Figure 8 As shown, the ploidy of soybean plant genome can be determined by the peak pattern detected by flow cytometry.

[0055] Specifically, when taking leaves, select the tenderest leaves at the top, and then take leaves the size of a fingernail and put them in a culture dish.

[0056] In some embodiments of the present invention, the soil in the seedling pot is composed of loess and nutrient soil in a volume ratio of 1:1; the nutrient soil is conventional commercially available nutrient soil well known in the art.

[0057] On the other hand, the method of inducing soybean polyploidy using colchicine provided by the present invention can be used in soybean polyploid breeding.

[0058] Example 1

[0059] S1. Select 50 soybean seeds with full grains and vigor, and soak the soybean seeds in a colchicine solution with a mass concentration of 0.05% for 6 hours in a dark environment without light to obtain soaked soybean seeds.

[0060] S2. After rinsing the soaked soybean seeds with running water for 1 hour, use newspaper and toilet paper to stack to make water storage paper, stack a layer of water storage paper on the water storage paper, arrange the rinsed soybean seeds neatly on the stacked water storage paper and then roll it up to prepare a seed bag.

[0061] S3, placing the seed package at room temperature, transplanting the emerged seeds into a seedling pot after the seeds germinate, and waiting for the seeds to grow true leaves to obtain soybean seedlings.

[0062] S4. Take the tenderest leaf from the top of each plant, put it into a culture dish the size of a fingernail, add 250 μL of CyStain UV Ploidy DAPI lysis buffer into the dish, and chop the leaf into a paste with a blade.

[0063] S5. Add 250 μL of lysis solution to the culture dish again, then rinse with ultrapure water, filter with a 50-mesh cell sieve into the flow tube in the flow cytometer for detection, and select soybean polyploid seedlings based on the results.

[0064] Example 2

[0065] S1. Select 50 soybean seeds with full grains and vigor, and soak the soybean seeds in a colchicine solution with a mass concentration of 0.10% for 6 hours in a dark environment without light to obtain soaked soybean seeds.

[0066] S2. After rinsing the soaked soybean seeds with running water for 1 hour, use newspaper and toilet paper to stack to make water storage paper, stack a layer of water storage paper on the water storage paper, arrange the rinsed soybean seeds neatly on the stacked water storage paper and then roll it up to prepare a seed bag.

[0067] S3, placing the seed package at room temperature, transplanting the emerged seeds into a seedling pot after the seeds germinate, and waiting for the seeds to grow true leaves to obtain soybean seedlings.

[0068] S4. Take the tenderest leaf from the top of each plant, put it into a culture dish the size of a fingernail, add 250 μL of CyStain UV Ploidy DAPI lysis buffer into the dish, and chop the leaf into a paste with a blade.

[0069] S5. Add 250 μL of lysis solution to the culture dish again, then rinse with ultrapure water, filter with a 50-mesh cell sieve into the flow tube in the flow cytometer for detection, and select soybean polyploid seedlings based on the results.

[0070] Example 3

[0071] S1. Select 50 soybean seeds with full grains and vigor, and soak the soybean seeds in a colchicine solution with a mass concentration of 0.15% for 6 hours in a dark environment without light to obtain soaked soybean seeds.

[0072] S2. After rinsing the soaked soybean seeds with running water for 1 hour, use newspaper and toilet paper to stack to make water storage paper, stack a layer of water storage paper on the water storage paper, arrange the rinsed soybean seeds neatly on the stacked water storage paper and then roll it up to prepare a seed bag.

[0073] S3, placing the seed package at room temperature, transplanting the emerged seeds into a seedling pot after the seeds germinate, and waiting for the seeds to grow true leaves to obtain soybean seedlings.

[0074] S4. Take the tenderest leaf from the top of each plant, put it into a culture dish the size of a fingernail, add 250 μL of CyStain UV Ploidy DAPI lysis buffer into the dish, and chop the leaf into a paste with a blade.

[0075] S5. Add 250 μL of lysis solution to the culture dish again, then rinse with ultrapure water, filter with a 50-mesh cell sieve into the flow tube in the flow cytometer for detection, and select soybean polyploid seedlings based on the results.

[0076] Example 4

[0077] S1. Select 50 soybean seeds with full grains and vigor, and soak the soybean seeds in a colchicine solution with a mass concentration of 0.2% for 6 hours in a dark environment without light to obtain soaked soybean seeds.

[0078] S2. After rinsing the soaked soybean seeds with running water for 1 hour, use newspaper and toilet paper to stack to make water storage paper, stack a layer of water storage paper on the water storage paper, arrange the rinsed soybean seeds neatly on the stacked water storage paper and then roll it up to prepare a seed bag.

[0079] S3, placing the seed package at room temperature, transplanting the emerged seeds into a seedling pot after the seeds germinate, and waiting for the seeds to grow true leaves to obtain soybean seedlings.

[0080] S4. Take the tenderest leaf from the top of each plant, put it into a culture dish the size of a fingernail, add 250 μL of CyStain UV Ploidy DAPI lysis buffer into the dish, and chop the leaf into a paste with a blade.

[0081] S5. Add 250 μL of lysis solution to the culture dish again, then rinse with ultrapure water, filter with a 50-mesh cell sieve into the flow tube in the flow cytometer for detection, and select soybean polyploid seedlings based on the results.

[0082] Example 5

[0083] S1. Select 50 soybean seeds with full grains and vigor, and soak the soybean seeds in a colchicine solution with a mass concentration of 0.05% for 12 hours in a dark environment without light to obtain soaked soybean seeds.

[0084] S2. After rinsing the soaked soybean seeds with running water for 1 hour, use newspaper and toilet paper to stack to make water storage paper, stack a layer of water storage paper on the water storage paper, arrange the rinsed soybean seeds neatly on the stacked water storage paper and then roll it up to prepare a seed bag.

[0085] S3, placing the seed package at room temperature, transplanting the emerged seeds into a seedling pot after the seeds germinate, and waiting for the seeds to grow true leaves to obtain soybean seedlings.

[0086] S4. Take the tenderest leaf from the top of each plant, put it into a culture dish the size of a fingernail, add 250 μL of CyStain UV Ploidy DAPI lysis buffer into the dish, and chop the leaf into a paste with a blade.

[0087] S5. Add 250 μL of lysis solution to the culture dish again, then rinse with ultrapure water, filter with a 50-mesh cell sieve into the flow tube in the flow cytometer for detection, and select soybean polyploid seedlings based on the results.

[0088] Example 6

[0089] S1. Select 50 soybean seeds with full grains and vigor, and soak the soybean seeds in a colchicine solution with a mass concentration of 0.10% for 12 hours in a dark environment without light to obtain soaked soybean seeds.

[0090] S2. After rinsing the soaked soybean seeds with running water for 1 hour, use newspaper and toilet paper to stack to make water storage paper, stack a layer of water storage paper on the water storage paper, arrange the rinsed soybean seeds neatly on the stacked water storage paper and then roll it up to prepare a seed bag.

[0091] S3, placing the seed package at room temperature, transplanting the emerged seeds into a seedling pot after the seeds germinate, and waiting for the seeds to grow true leaves to obtain soybean seedlings.

[0092] S4. Take the tenderest leaf from the top of each plant, put it into a culture dish the size of a fingernail, add 250 μL of CyStain UV Ploidy DAPI lysis buffer into the dish, and chop the leaf into a paste with a blade.

[0093] S5. Add 250 μL of lysis solution to the culture dish again, then rinse with ultrapure water, filter with a 50-mesh cell sieve into the flow tube in the flow cytometer for detection, and select soybean polyploid seedlings based on the results.

[0094] Example 7

[0095] S1. Select 50 soybean seeds with full grains and vigor, and soak the soybean seeds in a colchicine solution with a mass concentration of 0.15% for 12 hours in a dark environment without light to obtain soaked soybean seeds.

[0096] S2. After rinsing the soaked soybean seeds with running water for 1 hour, use newspaper and toilet paper to stack to make water storage paper, stack a layer of water storage paper on the water storage paper, arrange the rinsed soybean seeds neatly on the stacked water storage paper and then roll it up to prepare a seed bag.

[0097] S3, placing the seed package at room temperature, transplanting the emerged seeds into a seedling pot after the seeds germinate, and waiting for the seeds to grow true leaves to obtain soybean seedlings.

[0098] S4. Take the tenderest leaf from the top of each plant, put it into a culture dish the size of a fingernail, add 250 μL of CyStain UV Ploidy DAPI lysis buffer into the dish, and chop the leaf into a paste with a blade.

[0099] S5. Add 250 μL of lysis solution to the culture dish again, then rinse with ultrapure water, filter with a 50-mesh cell sieve into the flow tube in the flow cytometer for detection, and select soybean polyploid seedlings based on the results.

[0100] Example 8

[0101] S1. Select 50 soybean seeds with full grains and vigor, and soak the soybean seeds in a colchicine solution with a mass concentration of 0.20% for 12 hours in a dark environment without light to obtain soaked soybean seeds.

[0102] S2. After rinsing the soaked soybean seeds with running water for 1 hour, use newspaper and toilet paper to stack to make water storage paper, stack a layer of water storage paper on the water storage paper, arrange the rinsed soybean seeds neatly on the stacked water storage paper and then roll it up to prepare a seed bag.

[0103] S3, placing the seed package at room temperature, transplanting the emerged seeds into a seedling pot after the seeds germinate, and waiting for the seeds to grow true leaves to obtain soybean seedlings.

[0104] S4. Take the tenderest leaf from the top of each plant, put it into a culture dish the size of a fingernail, add 250 μL of CyStain UV Ploidy DAPI lysis buffer into the dish, and chop the leaf into a paste with a blade.

[0105] S5. Add 250 μL of lysis solution to the culture dish again, then rinse with ultrapure water, filter with a 50-mesh cell sieve into the flow tube in the flow cytometer for detection, and select soybean polyploid seedlings based on the results.

[0106] Comparative Example 1

[0107] S1. Select 50 soybean seeds with full grains and vigor, and soak the soybean seeds in pure water for 6 hours in a dark environment without light to obtain soaked soybean seeds.

[0108] S2. After rinsing the soaked soybean seeds with running water for 1 hour, use newspaper and toilet paper to stack to make water storage paper, stack a layer of water storage paper on the water storage paper, arrange the rinsed soybean seeds neatly on the stacked water storage paper and then roll it up to prepare a seed bag.

[0109] S3, placing the seed package at room temperature, transplanting the emerged seeds into a seedling pot after the seeds germinate, and waiting for the seeds to grow true leaves to obtain soybean seedlings.

[0110] S4. Take the tenderest leaf from the top of each plant, put it into a culture dish the size of a fingernail, add 250 μL of CyStain UV Ploidy DAPI lysis buffer into the dish, and chop the leaf into a paste with a blade.

[0111] S5. Add 250 μL of lysis solution to the culture dish again, then rinse with ultrapure water, filter with a 50-mesh cell sieve into the flow tube in the flow cytometer for detection, and select soybean polyploid seedlings based on the results.

[0112] Comparative Example 2

[0113] S1. Select 50 soybean seeds with full grains and vigor, and soak the soybean seeds in pure water for 6 hours in a dark environment without light to obtain soaked soybean seeds.

[0114] S2. After rinsing the soaked soybean seeds with running water for 1 hour, the rinsed soybean seeds are placed in a seed tray hole with a hole depth of 9 cm, an upper diameter of 4.8 cm, and a lower diameter of 1.8 cm.

[0115] S3. After germination, the seeds are transplanted into a seedling pot and waited for true leaves to grow to obtain soybean seedlings.

[0116] S4. Take the tenderest leaves from the top of each plant, tear them into fingernail-sized pieces and place them in a culture dish. Add 250 μL of CyStain UV Ploidy DAPI lysis buffer to the dish and chop the leaves into a paste with a blade.

[0117] S5. Add 250 μL of lysis solution to the culture dish again, then rinse with ultrapure water, filter with a 50-mesh cell sieve into the flow tube in the flow cytometer for detection, and select soybean polyploid seedlings based on the results.

[0118] Comparative Example 3

[0119] S1. Select 50 soybean seeds with full grains and vigor, and soak the soybean seeds in a colchicine solution with a mass concentration of 0.05% for 6 hours in a dark environment without light to obtain soaked soybean seeds.

[0120] S2. After rinsing the soaked soybean seeds with running water for 1 hour, the rinsed soybean seeds are placed in a seed tray hole with a hole depth of 9 cm, an upper diameter of 4.8 cm, and a lower diameter of 1.8 cm.

[0121] S3. After germination, the seeds are transplanted into a seedling pot and waited for true leaves to grow to obtain soybean seedlings.

[0122] S4. Take the tenderest leaf from the top of each plant, put it into a culture dish the size of a fingernail, add 250 μL of CyStain UV Ploidy DAPI lysis buffer into the dish, and chop the leaf into a paste with a blade.

[0123] S5. Add 250 μL of lysis solution to the culture dish again, then rinse with ultrapure water, filter with a 50-mesh cell sieve into the flow tube in the flow cytometer for detection, and select soybean polyploid seedlings based on the results.

[0124] Comparative Example 4

[0125] S1. Select 50 soybean seeds with full grains and vigor, and soak the soybean seeds in a colchicine solution with a mass concentration of 0.05% for 12 hours in a dark environment without light to obtain soaked soybean seeds.

[0126] S2. After rinsing the soaked soybean seeds with running water for 1 hour, the rinsed soybean seeds are placed in a seed tray hole with a hole depth of 9 cm, an upper diameter of 4.8 cm, and a lower diameter of 1.8 cm.

[0127] S3. After germination, the seeds are transplanted into a seedling pot and waited for true leaves to grow to obtain soybean seedlings.

[0128] S4. Take the tenderest leaf from the top of each plant, put it into a culture dish the size of a fingernail, add 250 μL of CyStain UV Ploidy DAPI lysis buffer into the dish, and chop the leaf into a paste with a blade.

[0129] S5. Add 250 μL of lysis solution to the culture dish again, then rinse with ultrapure water, filter with a 50-mesh cell sieve into the flow tube in the flow cytometer for detection, and select soybean polyploid seedlings based on the results.

[0130] Effect test

[0131] The seed survival rates in Examples 1-8 and Comparative Examples 1-4 were statistically analyzed, and the specific results are shown in Table 1 below:

[0132] Table 1 Statistics of seedling rate under different colchicine treatment conditions

[0133]

[0134] By comparing the data of Control Example 3 and Example 1, it can be seen that when the mass concentration of colchicine is 0.05% and the soaking time is 6 hours, the survival rate of the plants in Control Example 3 using plug tray germination is 40%, while the survival rate of the plants in Example 1 using seed bag germination is 50%.

[0135] By comparing the data of Control Example 4 and Example 5, it can be seen that when the mass concentration of colchicine is 0.05% and the soaking time is 12 hours, the survival rate of the plants in Control Example 4 using plug tray germination is 10%, while the survival rate of the plants in Example 5 using seed bag germination is 46%.

[0136] In summary, the seed-encapsulating seedling raising method of the present invention can significantly improve the plant survival rate of soybean seeds treated with colchicine.

[0137] The doubling success rates in Examples 1-8 were statistically analyzed, and the specific results are shown in Table 2 below:

[0138] Table 2 Statistics of doubling success rate under different colchicine treatment conditions

[0139]

[0140] The results showed that different treatments had different doubling success rates. The tetraploid induction rates of 0.05% colchicine soaking for 12h (0.05%_12h) and 0.20% colchicine soaking for 12h (0.2%_12h) were the highest, both 4.00%, but the chimera induction rate of 0.2%_12h treatment was 2%, so the possibility of obtaining tetraploids in 0.2%_12h treatment was the highest; in addition, the survival rate of soybean plants in 0.2%_12h treatment was 34%, lower than the survival rate of 46% in 0.05%_12h treatment, which made the workload and cost of ploidy identification of surviving plants in 0.2%_12h treatment less than that in 0.05%_12h treatment. Therefore, considering the cost of ploidy identification in the later stage, 0.2%_12h treatment had the highest doubling efficiency and the lowest cost.

[0141] The surviving soybean plants were subsequently cultured and their self-pollinated seeds were harvested. The self-pollinated seeds of diploid and tetraploid soybeans were sown, and the images of the morphology, leaves, petals and harvested soybean seeds of diploid and tetraploid plants at the same period were collected. The protein content of diploid and tetraploid soybeans was tested, and the results are as follows:

[0142] like Figure 2-6 As shown, compared with diploid soybean plants, tetraploid plants have greater differences in plant shape and increased genetic diversity; the leaves have become rounded, and some leaves have become four-petal compound leaves and five-petal compound leaves; the petals and seeds of tetraploid plants have become larger; the quality of tetraploid plants also changes: the protein content and water-soluble protein content increase.

[0143] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for inducing soybean polyploidy using colchicine, characterized in that: The following steps are involved: Selecting soybean seeds, placing the soybean seeds in a colchicine solution for soaking, and obtaining soaked soybean seeds; The soaked soybean seeds are post-processed, and then the soybean seeds are wrapped with water storage paper to obtain seed bags; The seed package is placed in a room temperature environment, and after the seeds germinate, the germinated seeds are transplanted into a seedling pot, and after the true leaves are grown, the surviving individual plants are identified and screened to obtain soybean polyploid seedlings.

2. The method for inducing soybean polyploidy using colchicine according to claim 1, characterized in that: The mass concentration of the colchicine solution is 0.05%-0.2%.

3. The method for inducing soybean polyploidy using colchicine according to claim 1, characterized in that: The soaking time is 6-12 hours, and the soaking environment is a dark environment without light.

4. The method for inducing soybean polyploidy using colchicine according to claim 1, characterized in that: The post-treatment is specifically to wrap the soaked soybean seeds in a mesh bag and rinse them with running water for 1 hour.

5. The method for inducing soybean polyploidy using colchicine according to claim 1, characterized in that: The water storage paper is formed by stacking newspaper and toilet paper. The seed bag is wrapped in the following way: stacking a layer of water storage paper on the water storage paper, arranging the soybean seeds neatly and then rolling them up.

6. The method for inducing soybean polyploidy using colchicine according to claim 1, characterized in that: The specific method of the identification and screening is: Take leaves from the surviving individual plants and place them in culture dishes, add 250 μL of lysis solution to the culture dishes, and chop the leaves into a paste with a blade; 250 μL of lysis solution was added to the culture dish again, followed by rinsing with ultrapure water, and filtered through a 50-mesh cell sieve into a flow tube in a flow cytometer for detection, and soybean polyploid seedlings were screened out based on the results.

7. The method for inducing soybean polyploidy using colchicine according to claim 1, characterized in that: The soil in the seedling pot is made of loess and nutrient soil in a volume ratio of 1:

1.

8. Use of the method for inducing soybean polyploidy using colchicine as described in any one of claims 1 to 7 in soybean polyploid breeding.

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