Polyploid breeding method for chromosome group doubling of wheat plants

Abstract

The invention discloses a polyploidy breeding method for wheat plant genome doubling, which belongs to the technical field of plant breeding, and comprises the following steps: treating germinated wheat for 30 days under the conditions of low temperature of 2-7 DEG C and 24-hour illumination, then transplanting into soil, culturing for 45-55 days under the conditions that the temperature is 20-25 DEG C, the light-dark culture period is 20-22 hours, and the light-dark culture period is 2-4 hours, the illumination intensity is 10000-20000 lx, and the culture time is 20-30 days. Wherein the ratio of the red light to the blue light is 3: 2, obtaining a large number of chromosome group doubled seeds until the wheat grains are mature, and realizing wheat plant chromosome group doubling. Experiments prove that the method is easy to operate, toxic reagents are avoided, the period is short, and the efficiency of obtaining wheat with doubled chromosome groups is high.

Description

Technical Field

[0001] The invention belongs to the technical field of plant breeding, and in particular relates to a polyploid breeding method for triticum plants by doubling their chromosome sets. Background Art

[0002] Given limited arable land, increasing the yield of grain crops is an effective and important measure. Cultivating new high-yield varieties is an important way to increase grain production in the future.

[0003] Polyploid breeding refers to the process of obtaining polyploid breeding material by doubling the chromosome set of cells through artificial mutagenesis or natural variation, with the goal of selecting superior varieties that meet human needs. Polyploid varieties developed through chromosome doubling often exhibit the following characteristics: 1) sturdy stems, with relatively large leaves, fruits, and seeds; 2) high metabolism and strong adaptability to the environment, which can expand the original variety's planting environment; 3) nutrient-rich content, with high levels of carbohydrates, vitamins, protein, and alkaloids; and 4) slow growth, delayed development, reduced respiration and transpiration, and enhanced resistance.

[0004] Currently, polyploid breeding projects often use colchicine or similar chemicals to treat germinating seeds or seedlings. Colchicine inhibits spindle formation during mitosis but does not affect chromosome replication, preventing the formation of two daughter cells and doubling the number of chromosomes. Colchicine, an alkaloid originally extracted from the autumn lily, is highly toxic and can cause plant death in severe cases. Currently, there is no antidote for clinical application. Using colchicine to induce chromosome doubling requires exploring the appropriate concentration and treatment time, which has certain thresholds. Even when treating plant seedlings at the optimal concentration and time, chromosome doubling efficiency is low, requiring multiple treatments. Furthermore, the polyploids obtained are often mosaics and often lack stable inheritance. Addressing the limitations of colchicine-induced chromosome doubling, improved induction methods are urgently needed.

[0005] Wheat is the world's largest staple food crop, with over one-third of the world's population relying on it as their staple food. Wheat belongs to the genus Triticeae in the Poaceae family. The most common common wheat is hexaploid, accounting for over 90% of the world's total wheat planting area. In addition to common wheat, the genus Triticeae also includes single-grain and two-grain wheat lines. Ensuring food security requires the development of new wheat varieties that are high-yielding, high-quality, and multi-resistant. In production, the development of new wheat varieties is largely based on conventional hybrid breeding techniques. Even with experienced breeders, developing an ideal new wheat variety often requires over a decade of effort. Typically, the probability of successfully breeding a wheat variety is as low as 5 / 1000, meaning only one suitable new variety can be developed from over 200 combinations. Efficient and scientific breeding methods are urgently needed to address these shortcomings in the Triticeae genus, particularly common wheat. Summary of the Invention

[0006] In order to make up for the shortcomings of the existing technology, the present invention provides a polyploid breeding method for doubling the chromosome set of wheat plants. After treatment with this method, the success rate of chromosome doubling in wheat can reach 40%-63.33%.

[0007] In order to achieve the above objectives, the present invention adopts the following technical solutions: As a first aspect of the present invention, a polyploid breeding method for chromosome doubling of a Triticum plant is provided, comprising the following steps: S1, low temperature and continuous light treatment: seeds were first germinated at room temperature, and then the germinated wheat seedlings were moved to a low temperature environment for vernalization treatment; S2, room temperature culture: The seedlings obtained in S1 are transferred to a greenhouse for further culture, and are treated with red and blue light to obtain polyploids.

[0008] Preferably, in step S1, the seeds are placed in a culture dish lined with filter paper, an appropriate amount of water is added, and the seeds are germinated at room temperature.

[0009] Preferably, in step S1, after germination for 2-5 days at room temperature, the wheat seedlings are moved to a low temperature environment for 24 hours of light treatment.

[0010] Preferably, in step S1, the temperature of the low-temperature environment is 2-7°C.

[0011] Preferably, in step S1, the vernalization treatment is performed for 25-30 days.

[0012] Preferably, in step S2, the greenhouse culture conditions are: temperature of 20-25°C, relative humidity of 60-70%, and culture time of 45-55 days.

[0013] Furthermore, in step S2, the greenhouse culture conditions are: the light-dark culture cycle is light culture for 20-22 hours and dark culture for 2-4 hours, and the light intensity is 10,000-20,000 lx.

[0014] Furthermore, in step S2, the greenhouse culture conditions are: the red light wavelength range is 625-740nm, and the blue light wavelength range is 440-475nm.

[0015] Furthermore, in step S2, the greenhouse culture conditions are: the red light: blue light intensity ratio range is 3:2.

[0016] In an embodiment of the present invention, in step S1, the seeds are placed in a culture dish lined with filter paper, an appropriate amount of water is added, and the seeds are germinated at room temperature. After germination at room temperature for 2-5 days, the wheat seedlings are moved to a low-temperature environment of 2-7°C for 24 hours of light treatment and vernalization treatment for 25-30 days; S2, room temperature culture: The seedlings obtained in S1 were transferred to a greenhouse for further culture for 45 to 55 days. The greenhouse conditions were as follows: temperature of 20-25°C, relative humidity of 60-70%, light-dark culture cycle of 20-22 hours of light culture and 2-4 hours of dark culture, light intensity of 10,000-20,000 lx, wherein the red light wavelength range was 625-740 nm, the blue light wavelength range was 440-475 nm, and the red light: blue light intensity ratio range was 3:2.

[0017] In one or more embodiments of the present invention, the Triticum plant includes common wheat.

[0018] The present invention achieves chromosome doubling of common wheat through low temperature (2-7°C) and long-term (22-24 hours) illumination, and this technical means can be used for polyploid breeding of the genus Triticum.

[0019] As a second aspect of the present invention, it provides the application of the method in polyploid breeding of common wheat.

[0020] The principle is analyzed as follows: in an appropriate low temperature and light environment, the genetic material replicates normally during the wheat mitosis process. Low temperature inhibits the formation of the spindle, and the chromosomes cannot be pulled to the two poles. The chromosomes remain in the center of the cell, causing the number of chromosomes in the cell to double. The treated wheat is then moved to a 22-24h long light environment to promote ear formation and quickly obtain offspring with doubled chromosomes.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides a polyploid breeding method for plants of the genus Triticum, and its application in common wheat. The present invention provides a new wheat variety with doubled chromosome sets by treating wheat with low temperature and continuous light. 2. The method of the present invention can produce a large number of seeds with increased volume and doubled chromosome sets, achieving double the chromosome set of common wheat; the wheat cultivated by the method of the present invention has reduced plant height and increased stem thickness, making it more resistant to lodging; the ear length and width of the wheat ears are increased, the length and width of the grains are increased, and the thousand-grain weight is increased, which can significantly increase wheat yield; 3. The method of the present invention greatly improves the success rate of wheat variety breeding and makes up for the shortcomings in the breeding of the genus Triticeae, especially common wheat. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0023] Figure 1: After 30 days of low temperature and continuous light treatment, cultivation under red and blue light reduced the plant height of common wheat, but increased the stem diameter of common wheat; among them, A is the photo of wheat under different treatments, B is the change of plant height under different treatments, and C is the change of stem diameter under different treatments.

[0024] Figure 2 : The growth period of common wheat was delayed by culturing under red and blue light after 30 days of low temperature and continuous light treatment.

[0025] Figure 3 : Effects of low temperature and continuous light treatment on the ear length and ear width of common wheat cultured under red and blue light after 30 days of treatment; A is the ear length under different treatments, and B is the ear width under different treatments.

[0026] Figure 4 : After 30 days of low temperature and continuous light treatment, the grain length of common wheat grains increased under red and blue light; A is a photo of grain length under different treatments, and B is a comparison of grain length data under different treatments.

[0027] Figure 5 : After 30 days of low temperature and continuous light treatment, the grain width of common wheat grains increased when cultured under red and blue light; A is a photo of grain width under different treatments, and B is a comparison of grain width data under different treatments.

[0028] Figure 6 : The thousand-grain weight of common wheat grains was increased by culturing under red and blue light after 30 days of low temperature and continuous light treatment.

[0029] Figure 7 : The number of chromosomes of common wheat doubled after 30 days of low temperature and continuous light treatment under red and blue light.

[0030] Figure 8 : The proportions of doubled common wheat (n=42) and chromosome-doubled materials (n=84) cultured under white light and red and blue light after 30 days of low temperature and continuous light treatment. DETAILED DESCRIPTION

[0031] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0032] The present invention utilizes low temperature and continuous light treatment for 30 days to explore its effect on the chromosome number of common wheat.

[0033] In an embodiment of the present invention, a polyploid breeding method for chromosome doubling of a Triticum plant is provided, comprising the following steps: S1, low temperature and continuous light treatment: seeds were placed in a culture dish lined with filter paper, added with appropriate amount of water, and germinated at room temperature; after germination at room temperature, the wheat seedlings were moved to a low temperature environment for vernalization treatment; In the embodiment of the present invention, the objects of the chromosome set doubling treatment are common wheat seedlings that have germinated for 2-5 days.

[0034] In the embodiment of the present invention, the germinated seedlings are treated at a low temperature of 2-7°C and 24 hours of light for 25-30 days.

[0035] S2, room temperature cultivation: The seedlings obtained in S1 are transferred to a greenhouse for further cultivation for 45-55 days.

[0036] In the embodiment of the present invention, the greenhouse conditions are as follows: temperature of 20-25°C, relative humidity of 60-70%, light and dark culture cycle of 20-22 hours of light culture and 2-4 hours of dark culture, light intensity of 10000-20000 lx, wherein the red light wavelength range is 625-740 nm, the blue light wavelength range is 440-475 nm, and the red light: blue light intensity ratio is 3:2.

[0037] Example 1: Low temperature and continuous light treatment for 30 days doubles the number of chromosomes in Aegilops tauschii (1) Overview of the test site and test materials The experimental site is located at Building B5, No. 3366, Longteng Road, Beijipo Street, Daiyue District, Tai'an City, Shandong Province.

[0038] Common wheat varieties: Jimai 22 (JM22), Shannong 981 (SN981).

[0039] (2) Experimental design The seeds of common wheat varieties were placed in a culture dish lined with filter paper, and appropriate amount of water was added to germinate at room temperature.

[0040] There are two treatments for common wheat: Treatment 1: control group (CK), no low-temperature light treatment; Treatment 2: Common wheat was treated with low temperature and continuous light for 30 days; after germination for 2 days at room temperature, the wheat seedlings were moved to a low temperature environment of 4℃ for 24 hours of light treatment and vernalization treatment for 30 days.

[0041] A randomized block design was adopted, with three replications for each treatment, and each replication consisted of four plastic pots (9×9×10 cm). Nine common wheat seedlings were transplanted into each pot and transferred to the greenhouse for further cultivation for 55 days. The greenhouse conditions were as follows: temperature of 20°C, relative humidity of 60%, light-dark cycle of 22 h of light cultivation and 2 h of dark cultivation, light intensity of 20,000 lx, red light wavelength of 650 nm, blue light wavelength of 450 nm, and red light: blue light = 3:2.

[0042] Example 2, The same treatment as in Example 1 was used, except that after germination for 2 days at room temperature, the wheat seedlings were moved to a low-temperature environment of 4°C for 24 hours of light treatment and vernalization treatment for 25 days.

[0043] Example 3, The same treatment as in Example 1 was used, except that the illumination condition for wheat cultivation after 30 days of vernalization was white light.

[0044] Test examples, measurement indicators and measurement methods The following test was conducted on the wheat at harvest time in Example 1: 1. Determination of plant height: Three replicates were taken for each treatment at the harvest stage. Ten common wheat plants were randomly selected from each replicate. The height from the base of the wheat to the top of the ear was measured with a ruler and the average value was calculated.

[0045] Determination of stem diameter: Three replicates were taken for each treatment at the harvest stage, and 10 common wheat plants were randomly selected from each replicate. The diameter at the middle position between the first and second internodes at the base of the stem was measured with a vernier caliper and recorded as the stem diameter of the common wheat. The average value was calculated.

[0046] The results are as follows Figure 1 As shown, compared with the wild-type control (WT), the wheat plant height of the chromosome-doubled wheat obtained by the method of Example 1 was significantly reduced ( Figure 1 A, B), but the stem diameter increased significantly ( Figure 1 A, C). It can be seen that after 30 days of low temperature and continuous light treatment, long-term light culture under red and blue light reduced wheat plant height but increased wheat stem diameter.

[0047] 2. Determination of growth period: Three replicates were taken for each treatment at the harvest period, and 10 common wheat plants were randomly selected from each replicate. The number of days between wheat sowing and entering the waxy stage was recorded as the growth period of wheat, and the average value was calculated.

[0048] The results are as follows Figure 2 As shown in the figure, the growth period of wheat with chromosome doubling (Dou) was significantly delayed compared with the wild-type control (WT). It can be seen that long-term light culture under red and blue light after 30 days of low temperature and continuous light treatment delayed the growth period of wheat.

[0049] 3. Determination of wheat ear length and width: Three replicates were taken for each treatment during the harvest period. Ten common wheat plants were randomly selected from each replicate. The length and width of the wheat ears were measured with a ruler, and the average values were calculated and recorded as the wheat ear length and ear width, respectively.

[0050] The results are as follows Figure 3 As shown in A and B, compared with the wild-type control (WT), the ear length and ear width of wheat ears with chromosome doubling (Dou) were significantly increased. It can be seen that long-term illumination culture under red and blue light increased the ear length and ear width of wheat ears after 30 days of low temperature and continuous light treatment.

[0051] 4. Determination of wheat grain length (GL): Using dried grains as the standard, five replicates were taken for each treatment. Ten grains were randomly selected from each replicate and arranged end to end. The total length was measured with a ruler, and then the average length of the grains was calculated and recorded as the wheat grain length.

[0052] The results are as follows Figure 4 As shown in A and B, the grain length of wheat grains with chromosome doubling (Dou) was significantly increased compared with the wild-type control (WT). It can be seen that long-term illumination culture under red and blue light increased the grain length of wheat grains after 30 days of low temperature and continuous light treatment.

[0053] 5. Determination of wheat grain width (GW): Take dried grains as the standard, take five replicates for each treatment, randomly select 10 grains and arrange them side by side, measure the total width with a ruler, and then calculate the average length of the grains, which is recorded as the wheat grain width.

[0054] The results are as follows Figure 5 As shown in A and B, the grain width of wheat grains with doubled chromosomes (Dou) was significantly increased compared with the wild-type control (WT). It can be seen that long-term illumination culture under red and blue light increased the grain width of wheat grains after 30 days of low-temperature continuous illumination treatment.

[0055] 6. Determination of thousand-kernel weight (TGW): Using oven-dried kernels as the standard, three replicates were taken for each treatment. Fifty kernels were randomly selected from each replicate and weighed. The average value was calculated and then converted to the thousand-kernel weight, which was recorded as the thousand-kernel weight of wheat kernels.

[0056] The results are as follows Figure 6 As shown in A and B, the thousand-grain weight of wheat grains with chromosome doubling (Dou) was significantly increased compared with the wild-type control (WT). It can be seen that long-term illumination culture under red and blue light increased the thousand-grain weight of wheat grains after 30 days of low temperature and continuous light treatment.

[0057] 7. Determination of Chromosome Number: Waxy wheat kernels were oven-dried at 30°C for 24 hours, placed in a Petri dish lined with filter paper, and an appropriate amount of water was added. Germination was allowed to proceed at room temperature. Two days after germination, actively growing root tips were collected for pressing. The root tips were fixed in Carnoy's fixative (anhydrous ethanol: glacial acetic acid = 3:1) for 3 hours, rinsed with distilled water, and then dissociated in dissociation solution (hydrochloric acid:anhydrous ethanol = 1:1) for 10 minutes. The root tips were then rinsed three times with distilled water and stained with Carbofuchsin for 1 hour. The root tips were pressed using conventional methods and photographed microscopically. The number of chromosomes in metaphase cells was counted.

[0058] The results are as follows Figure 7 As shown in Figures A and B, the wild-type control (WT) has 42 chromosomes, while the chromosome-doubled wheat (Dou) has 84 chromosomes. This indicates that long-term illumination under red and blue light after 30 days of low-temperature continuous illumination can double the number of chromosomes in common wheat.

[0059] 8. Effect of white light on wheat chromosome doubling: After 30 days of low-temperature continuous light treatment, common wheat was transferred to white light (Example 3) or red-blue light (Example 1) for cultivation. After 55 days, the waxy wheat grains were harvested, oven-dried at 30°C for 24 hours, placed in a Petri dish lined with filter paper, and then added with an appropriate amount of water for germination at room temperature. After two days of germination, actively growing root tips were collected for compression experiments to count the number of chromosomes in the root tip cells.

[0060] The results are as follows Figure 8 As shown in the data, the number of chromosomes of wheat cultured under white light was 42, while the proportion of JM22 with doubled chromosome number (n=84) in wheat cultured under red and blue light was 63.33%; the proportion of SN981 wheat chromosome number doubling was 40%. It can be seen that long-term light culture under red and blue light after 30 days of low temperature continuous light treatment can increase the proportion of wheat chromosome doubling.

[0061] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A polyploid breeding method for triticum plants by chromosome doubling, characterized in that: The steps include: S1, low temperature and continuous light treatment: first germinate the seeds at room temperature, and then perform vernalization treatment on the germinating wheat seedlings, specifically: move them to a low temperature environment of 2-7°C and place them under 24-hour light treatment; S2, room temperature culture: The seedlings obtained in S1 are transferred to a greenhouse for further culture, and are treated with red and blue light to obtain polyploids.

2. The polyploid breeding method for triticum plants with chromosome doubling according to claim 1, characterized in that: In step S1, the seeds are placed in a culture dish lined with filter paper, water is added, and the seeds are germinated at room temperature.

3. The polyploid breeding method for triticum plants with chromosome doubling according to claim 1, characterized in that: In step S1, after germination for 2-5 days at room temperature, the wheat seedlings are moved to a low temperature environment for 24 hours of light treatment.

4. The polyploid breeding method for triticum plants with chromosome doubling according to claim 1, characterized in that: In step S1, the vernalization treatment is carried out for 25-30 days.

5. The polyploid breeding method for triticum plants with chromosome doubling according to claim 1, characterized in that: In step S2, the greenhouse culture conditions are: temperature of 20-25°C, relative humidity of 60-70%, and culture time of 45-55 days.

6. The polyploid breeding method for triticum plants with chromosome doubling according to claim 1, characterized in that: In step S2, the greenhouse culture conditions are: the light-dark culture cycle is light culture for 20-22 hours and dark culture for 2-4 hours, and the light intensity is 10,000-20,000 lx.

7. The polyploid breeding method for triticum plants with chromosome doubling according to claim 1, characterized in that: In step S2, the greenhouse culture conditions are: the red light wavelength range is 625-740nm, and the blue light wavelength range is 440-475nm.

8. The polyploid breeding method for triticum plants with chromosome doubling according to claim 1, characterized in that: In step S2, the greenhouse culture conditions are: the red light: blue light intensity ratio is 3:

2.

9. The polyploid breeding method for chromosome doubling of Triticum plants according to claim 1, characterized in that: The Triticum plant includes common wheat.

10. Use of the method according to any one of claims 1 to 9 in polyploid breeding of common wheat.

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