Method for accelerating medicago sativa l. breeding via photoperiod induction

Abstract

Provided in the present application is a method for accelerating Medicago sativa L. breeding by utilizing photoperiod induction. In the method, the flowering of seed seedlings of Medicago sativa L. is induced by using the following conditions: sunshine duration: 20-22 hours; light quality ratio: white light, blue light, red light, and far-red light; light intensity: 10,000-40,000 lx; and temperature: 23-26 degrees Celsius. According to the method of the present application, the spatiotemporal constraints in Medicago sativa L. breeding can be broken, and 4-6 generations of seeds are harvested within one year. The method has good application prospects in Medicago sativa L. breeding research and production.

Description

A method for accelerating alfalfa breeding using photoperiod induction Technical Field

[0001] The present application belongs to the field of plant breeding. Specifically, the present application provides a method for accelerating alfalfa breeding by utilizing photoperiod induction. Background Art

[0002] Alfalfa (Medicago sativa L.) is an autotetraploid, insect-pollinated, cross-pollinated perennial legume forage grass. Rich in nutrients, high yield, and palatable, it earns the nickname "King of Forage Grasses." In the field, flowering and harvesting times vary depending on factors such as location, climate, planting management, and variety. Generally, spring-sown alfalfa takes four to six months to mature, while autumn-sown alfalfa takes even longer. The progress of alfalfa breeding in my country and research on flowering, fertilization, and fruiting are limited by time and space.

[0003] Photoperiod refers to the alternating lengths of light and dark periods during the diurnal cycle. Photoperiodism in plants refers to the response of plants to the length of day and night (Liu Li, 2004). Alfalfa is a long-day plant, and long-day conditions promote early flowering (Chen Xiang, 2016). Light quality has a positive impact on plant flowering and fruiting. Studies have found that red, blue, and far-red light can promote early flowering (Sun Miao, 2023; Wu Xiaoxiao, 2023).

[0004] Alfalfa seed breeding has always relied on natural field conditions, which has limited the breeding process of alfalfa and the research on flowering, fertilization and fruiting in my country. Currently, there is little research on induced flowering of alfalfa. Summary of the Invention

[0005] This application utilizes long days and different light qualities to promote early flowering of alfalfa, in order to obtain better conditions, establish an alfalfa induction flowering and early fruiting system, accelerate the alfalfa breeding process, and promote the generation and reproduction of alfalfa seeds.

[0006] In one aspect, the present application provides a method for accelerating alfalfa breeding using photoperiod induction, wherein the following conditions are used to induce rapid flowering of alfalfa seedlings:

[0007] Daylight hours: 20-22 hours;

[0008] Light quality ratio: white light, blue light, red light, far-infrared light;

[0009] Light intensity: 10000-40000lx;

[0010] Temperature: 23-26 degrees Celsius.

[0011] Furthermore, the sunshine duration is 22 hours.

[0012] Further, the temperature is 26 degrees Celsius.

[0013] Furthermore, the light intensity is 11000-32000lx.

[0014] Furthermore, the light quality ratio is selected from one of the following (1)-(3):

[0015] (1) 450-465nm wavelength light: 34.9%, 450-480nm wavelength light: 51.53%, 615-650nm wavelength light: 13.2%, 730nm wavelength light: 0.27%;

[0016] (2) 450-465nm wavelength light: 93.38%, 450-480nm wavelength light: 0.42%, 615-650nm wavelength light: 4.75%, 730nm wavelength light: 1.45%;

[0017] (3) 380-399nm wavelength light: 0.06%, 400-499nm wavelength light: 29.28%, 500-599nm wavelength light: 15.38%, 600-699nm wavelength light: 44.81%, 700-780nm wavelength light: 10.47%.

[0018] Furthermore, the method uses the following conditions to induce rapid flowering of alfalfa seedlings:

[0019] Sunshine duration: 22 hours;

[0020] Light quality ratio: 450-465nm wavelength light: 93.38%, 450-480nm wavelength light: 0.42%, 615-650nm wavelength light: 4.75%, 730nm wavelength light: 1.45%;

[0021] Light intensity: 11780lx;

[0022] Temperature: 26 degrees Celsius.

[0023] Furthermore, the flowering induction treatment time of alfalfa seedlings is 18-24 days.

[0024] Furthermore, the flowering treatment time of the alfalfa seedlings was 22 days.

[0025] Furthermore, the alfalfa variety is selected from Zhongnong No. 1, Zhongmu No. 1, Avia or Knight No. 2, and the autumn dormancy level range is 2.4-8.

[0026] In another aspect, the present application provides the application of the above method in accelerating the generation and propagation of alfalfa. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 shows the plant height of different plant materials at day 0 of induction (left) and the flowering induction rate under various culture conditions.

[0028] Figure 2 shows that the seedlings of Zhongnong No. 1 (ZN-A) germinated and grown for 45 days under 14hWL conditions were induced to flower on 22 days under 22hFCL1 and 22hFCL2 conditions, while no flower was produced under 14hWL as the control condition.

[0029] FIG3 shows that no flower bud differentiation was observed when the plant materials were cultured for 5 days under the control conditions of 14 hWL in the same period.

[0030] Figure 4 shows that under the four induction conditions, ZM-2 flower buds appeared on the 16th day and flowered on the 20-23th day.

[0031] Figure 5 shows that Avia seedlings (AWY-A) flowered on day 29 of induction, Knight 2 seedlings (QS2-A) and Zhongmu No. 1 seedlings (ZM-A) had flower buds on day 29 of induction, and flowered on day 33 of induction.

[0032] Figure 6 shows that ZM-2 induces flower buds to appear on day 16 and flowers to bloom on day 22; ZM-1 induces flower buds to appear on day 21 and flowers to bloom on day 29; and ZM-A induces flower buds to appear on day 29 and flowers to bloom on day 33.

[0033] Figure 7 shows that the seedlings of Zhongmu No. 1 single plant A, which was cut to grow to about 20 cm (ZM-1), and the seedlings of Zhongmu No. 1 single plant B, which was cut to grow to about 20 cm (ZM-3), did not bloom after induced under 14hWL conditions for 30 days. They were then directly induced under 22hFCL2 conditions. ZM-3 bloomed after 16 days of induction, and ZM-1 bloomed after 22 days of induction.

[0034] FIG8 shows the changes in the single A cutting seedlings of Zhongmu No. 1 (ZM-1) after pollination under the induction condition of 22hFCL2. DETAILED DESCRIPTION

[0035] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to be limiting. These examples are for illustrative purposes only and in no way limit the scope of protection of the present invention.

[0036] Example 1 Different induction conditions promote early flowering of alfalfa

[0037] This experiment set up six culture conditions (Table 1) and selected seven different alfalfa plant materials for induction (Table 2). The number of seedlings induced to flower under different culture conditions is shown in Table 3. The flowering induction rate ranged from 75% to 100%. Seedlings of about 20 cm were selected for induction for all varieties (Figure 1). Among them, the Zhongnong No. 1 seedlings (ZN-A) germinated and grew for 45 days under 14hWL conditions and flowered after 22 days under 22hFCL1 and 22hFCL2 conditions. At the same time, they did not flower under 14hWL as the control condition (Figure 2). To eliminate the suspicion that late flowering under 14hWL conditions was rather than non-flowering, the plant materials were cultured for an additional 5 days under the 14hWL control condition during the same period, and no flower bud differentiation was found (Figure 3). This shows that 14h white light conditions cannot induce alfalfa to flower, while different ratios of 22h white light, blue light, red light, and far-red light can promote early flowering of alfalfa.

[0038] On this basis, we selected seedlings (ZM-2) of about 20 cm tall from single-plant A of Zhongmu No. 1 and compared the effects of different 22-hour light quality ratios on the induction of flowering in alfalfa. We found that under the conditions of 22hFCL2, 22hFCL3, 22hFCL4, and 22hFCL5, ZM-2 could flower in the non-flowering season. Although flower buds appeared in ZM-2 at 16 days and flowered at 20-23 days under all four induction conditions (Figure 4), flowering was relatively early under 22hFCL2 and 22hFCL4, and the flowering time consistency was relatively high among the same batch of seedlings, and the plants grew relatively robustly. However, flowering was relatively late under 22hFCL3 and 22hFCL5, and the flowering time consistency among the same batch of seedlings was relatively poor, with the maximum flowering time difference of 7 days within the same batch of seedlings.

[0039] In summary, 22hFCL1, 22hFCL2, and 22hFCL4 were superior in inducing alfalfa flowering among the six induction conditions, inducing flowering within 20-22 days, with relatively high consistency in flowering time and robust plant growth. In addition to white light, all three conditions included blue, red, and far-red light, with either red or blue light accounting for a significant proportion, suggesting that red and blue light may be important light sources for inducing alfalfa flowering.

[0040] Table 1. Six culture conditions

[0041] Note: The measurement was performed using a DELIXI DLY-1802 illuminometer at a distance of 5 cm under the light without any obstruction by plants.

[0042] Table 2. Plant material

[0043] Table 3. Statistics of the number of induced flowering seedlings

[0044] Example 2: Flowering induction of seedlings of different varieties

[0045] Under the induction conditions of 22hFCL2, seedlings of about 20 cm in low, medium, and high fall dormancy levels were selected, and they were induced to flower, namely QS2-A (Knight 2, 2.4), ZM-A (Zhongmu No. 1, 3), and AWY-A (Aweiya, 8). It was found that Aweiya seedlings (AWY-A) flowered on 29 days after induction, and flower buds appeared on 29 days after induction of Knight 2 seedlings (QS2-A) and Zhongmu No. 1 seedlings (ZM-A), and flowered on 33 days after induction (Figure 5). This shows that seedlings of about 20 cm of different varieties can all flower under the induction conditions of 22hFCL2.

[0046] Example 3: Flowering induction of seedlings of different growth ages

[0047] Under 22hFCL2 induction conditions, Zhongmu No. 1 seedlings (ZM-A), annual cuttings of Zhongmu No. 1 single plant A (ZM-1), and single plant A cuttings (ZM-2) were induced to flower. Flower buds appeared on ZM-2 after 16 days of induction and flowered on 22 days; flower buds appeared on ZM-1 after 21 days of induction and flowered on 29 days; and flower buds appeared on ZM-A after 29 days of induction and flowered on 33 days (Figure 6). Flowering occurred earlier in the cuttings than in the seedlings, and earlier in the cuttings than in the seedlings.

[0048] Seedlings treated in Example 4 were induced to flower

[0049] Seedlings of Zhongmu No. 1 plant A, cut to approximately 20 cm in length (ZM-1), and seedlings of Zhongmu No. 1 plant B, cut to approximately 20 cm in length (ZM-3), were induced under 14-hour WL conditions for 30 days without flowering. They were then directly induced under 22-hour FCL2 conditions. ZM-3 flowered after 16 days of induction, and ZM-1 flowered after 22 days of induction (Figure 7). This indicates that large seedlings grown normally during the non-flowering season can still flower when directly induced, indicating that the biomass conditions for inducing flowering vary across a range of plants. Whether directly induced at 20 cm or continued growing after 20 cm before induction, the biomass conditions for inducing flowering were achieved.

[0050] Example 5 Pollination and Fruiting Time under 22hFCL2 Conditions

[0051] Under the induction condition of 22hFCL2, the single-plant A cutting seedlings of Zhongmu No. 1 (ZM-1) were induced to flower on day 29. After flowering, they were bagged for pollination and fruiting. It was found that pods began to form 5 days after pollination. The pods showed a spiral upward growth from bottom to top. At this time, the stigma and style at the top of the pods could be clearly observed to gradually wilt and lose water. The pods completed the upward spiral growth (elongation growth) 7 days after pollination. The color of the pods was light green. At this time, the style and stigma at the top of the pods did not disappear, but showed a more wilted and dehydrated state. The pods were full and plump on day 15 of pollination. The pods began to turn yellow and brown on day 24 of pollination. At this time, the stigma and style at the top of the pods also began to turn brown and shriveled. The pods became shriveled and lost water on day 27 of pollination. The pods matured on day 33 of pollination, and the brown-yellow seeds could be harvested. At this time, the wrinkled, wilted and shriveled styles and stigmas at the top of the pods could still be observed (Figure 8).

[0052] In summary, the 22hFCL1, 22hFCL2, and 22hFCL4 conditions performed better in inducing flowering time in alfalfa, with durations ranging from 20 to 22 days, with no significant differences. All three induction conditions shared the common characteristic of including four colors of light (white, red, blue, and far-red). Compared to 22hFCL1 and 22hFCL4, seedlings induced to flower under 22hFCL2 developed more branches and new shoots. Therefore, 22hFCL2 was selected for experimental verification of inducing flowering in seedlings of different varieties and inducing flowering and pollination and fruiting in seedlings of different ages. The results showed that under 22hFCL2, seedlings approximately 20 cm in length could be induced to flower in 22 to 33 days, and mature seeds could be harvested around 33 days, for a total of 55 to 66 days to complete a single seed generation. Seeds germinated under 14hWL conditions grow for about 45 days to produce seedlings approximately 20 cm in length. Induced under 22hFCL2 conditions flower in 22-33 days, for a total of 67-78 days from seed to induced flowering. Therefore, using 22hFCL2 conditions can overcome the temporal and spatial limitations of alfalfa breeding, enabling the multi-generational propagation of alfalfa seeds, yielding 4-6 generations of seeds per year.

Claims

1. A method for accelerating alfalfa breeding by photoperiod induction, characterized in that: The method uses the following conditions to induce alfalfa seedlings to bloom quickly: Daylight hours: 20-22 hours; Light quality ratio: white light, blue light, red light, far-infrared light; Light intensity: 10000-40000lx; Temperature: 23-26 degrees Celsius.

2. The method according to claim 1, wherein the sunshine duration is 22 hours.

3. The method of claim 1, wherein the temperature is 26 degrees Celsius.

4. The method according to claim 1, wherein the light intensity is 11000-32000 lx.

5. The method according to claim 1, wherein the light quality ratio is selected from one of the following (1) to (3): (1) 450-465nm wavelength light: 34.9%, 450-480nm wavelength light: 51.53%, 615-650nm wavelength light: 13.2%, 730nm wavelength light: 0.27%; (2) 450-465nm wavelength light: 93.38%, 450-480nm wavelength light: 0.42%, 615-650nm wavelength light: 4.75%, 730nm wavelength light: 1.45%; (3) 380-399nm wavelength light: 0.06%, 400-499nm wavelength light: 29.28%, 500-599nm wavelength light: 15.38%, 600-699nm wavelength light: 44.81%, 700-780nm wavelength light: 10.47%.

6. The method according to claim 1, wherein the method uses the following conditions to induce rapid flowering of alfalfa seedlings: daylight hours: 22 hours; Light quality ratio: 450-465nm wavelength light: 93.38%, 450-480nm wavelength light: 0.42%, 615-650nm wavelength light: 4.75%, 730nm wavelength light: 1.45%; Light intensity: 11780lx; Temperature: 26 degrees Celsius.

7. The method according to any one of claims 1 to 6, wherein the flowering treatment time of the alfalfa seedlings is 18 to 24 days.

8. The method according to claim 7, wherein the flowering treatment time of the alfalfa seedlings is 22 days.

9. The method according to any one of claims 1 to 8, wherein the alfalfa variety is selected from alfalfa varieties such as Zhongnong No. 1, Zhongmu No. 1, Avia, and Knight No. 2, and the fall dormancy level ranges from 2.4 to 8.

10. Use of the method according to any one of claims 1 to 9 in accelerated generation propagation of alfalfa.

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